bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphoproteins associated with cyclic nucleotide stimulation of ciliary motility in Paramecium
Authors: Bonini Nancy M. , Nelson David L.
Published: April 2021

Permeabilized, MgATP-reactivated cells of Paramecium (models) respond to cyclic AMP and cyclic GMP by increasing forward swimming speed. In association with the motile response, cyclic AMP and 8-bromo-cyclic GMP (8-Br-cyclic GMP) stimulated protein phosphorylation. Cyclic AMP addition to permeabilized cells reproducibly stimulated the phosphorylation of 10 proteins, ranging in molecular weight from 15 to 110K (K=103Mr). 8-Br-cyclic GMP, which selectively activates the cyclic GMP-dependent protein kinase of Paramecium, stimulated the phosphorylation of a subset of the proteins phosphorylated by cyclic AMP. Ca2+ addition caused backward swimming and stimulated the phosphorylation of four substrates, including a 25K target that may also be phosphorylated in response to cyclic nucleotide addition. Ba2+ and Sr2+ also induced backward swimming, but did not cause detectable phosphorylation.

To identify ciliary targets of cyclic nucleotidedependent protein kinase activity, permeabilized cells were deciliated following reactivation of motility with Mg-[γ-32P]ATP in the presence or absence of cyclic nucleotide. Soluble proteins of the decilIation supernatant were enriched in 15 cyclic AMP-stimulated phosphoproteins, ranging in molecular weight from 15 to 95K. Most of the ciliary substrates were axonemal and could be released by high salt solution. A 29K protein that copurified in sucrose gradients with the 22 S dynein, and a high molecular weight protein (>З00K) in the 19 S region were phosphorylated when cyclic AMP was added to permeabilized, motile cells. These data suggest that regulation of ciliary motility by cyclic AMP may include phosphorylation of dynein-associated proteins.
Binding of exocytic vesicles from MDCK cells to microtubules in vitro
Authors: Sluijs Peter Van Der , Bennett Mark K. , Antony Claude , Simons Kai , Kreis Thomas E.
Published: April 2021

Microtubules have been implicated in the transport of vesicles carrying newly synthesized proteins from the trans-Golgi network (TGN) to the cell surface. We have established a quantitative in vitro binding assay to investigate the putative interaction between these exocytic carrier vesicles and the microtubules at the molecular level. TGN-derived exocytic carrier vesicles, labeled with C6NBD-cera-mide metabolites or viral glycoproteins, were obtained from polarized filter-grown MDCK II cells by perforation of the apical membrane with a nitrocellulose filter. These exocytic vesicles were incubated with taxol-polymerized tubulin and cytosol, layered on top of a 30% sucrose cushion and subjected to centrifugation. Quantitation of vesicles co-sedimenting with microtubules was done by measuring NBD-fluorescence of viral glycoproteins in the pellet and supernatant fractions. About 25% of the label sedimented through the cushion in the presence of microtubules and cytosol. Both apically and basolaterally targetted carrier vesicles containing influenza virus HA2 or vesicular stomatitis virus G protein, respectively, associated with the microtubules. Only 2-5% NBD-fluorescence was obtained in the pellet when no cytosol or microtubules were added to the vesicles. Negative-stain electron microscopy of resuspended pellets showed distinct microtubule-vesicle complexes. Heat inactivation or treatment of cytosol with N-ethylmaleimide (NEM), or trypsinization of vesicles inhibited the binding of vesicles to microtubules. Furthermore, coating of microtubules with brain microtubule-associated proteins abolished binding. These data suggest that NEM-sensitive cytosolic proteins are required for microtubule-vesicle association, and that the vesicles are bound via trypsin-sensitive receptor proteins on their surface.
Narrow linear strips of adhesive substratum are powerful inducers of both growth and total focal contact area
Authors: O’Neill Charles , Jordan Peter , Riddle Peter , Ireland Grenham
Published: April 2021

The stimulus to growth that occurs when cells attach to the substratum can be studied with small adhesive islands. Large numbers of these islands can be grouped together into arrays made up of various sizes, and the response of cells to incubation on these arrays allows the anchorage stimulus to be measured. Past work has shown that single isolated cells can be stimulated to proliferate under these circumstances quite as freely as they do in ordinary cultures, and that the maximum response is given by islands whose size is less than 5000μm2.

This anchorage stimulus might be mediated by the cytoskeleton, which assembles rapidly around the points of attachment to the substratum. One possible approach to testing this hypothesis is to expose cells to islands of different shapes, and to search for common factors among the different arrangements of the cytoskeleton that these different islands cause.

Circular islands induced a relatively disordered arrangement of actin fibres. The fibres were attached at one end to foci of vinculin, which sometimes became arranged in a ring around the margin of the island. Triangular islands showed a more orderly arrangement of actin, in three bands parallel to the sides. In this case, the vinculin accumulated at the apices. Long islands only 3μm wide could also provide effective attachment for the cells. In this shape the actin accumulated in two bands 2 μm or more apart and up to 5 μm high, and the vinculin similarly collected in parallel interrupted bands along the margins of the island. The number of vinculin foci differed on these three different island shapes, and the total area of vinculin was more than three times greater on long islands than on circles or triangles of the same size.

Despite these differences, all three different shapes of island were capable of inducing up to 100μm2 of vinculin foci in each cell. Round and triangular islands induced this maximum amount of vinculin when their size was 5000 μm2. Linear islands induced the same amount when they were only 1000 μm2.

The effect of different shapes on total vinculin focal area was paralleled by their effects on growth. All three shapes could support a similar amount of proliferation. Round and triangular islands induced the maximum amount of proliferation when they were 5000 μm2 in area, and linear islands when they were only 1000μm2. Similarly, linear islands induced as many cells to undergo DNA synthesis as did circles more than twice as large.

These experiments support the idea that focal contacts might act as tranducers of the anchorage stimulus to growth. They show that total focal contact area is consistently and quantitatively related to growth, and suggest that the arrangement of the foci or their number may have less importance. Island substrata of different shapes offer a new approach towards the understanding of the involvement of the cytoskeleton in the mechanism of growth regulation.
Higher-order chromosome structure in yeast
Authors: Yanagida Mitsuhiro
Published: April 2021

In this commentary I shall discuss recent findings on higher-order chromosome structure in yeast, a unicellular eukaryote. There are two kinds of yeast under current intensive investigation: namely, the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. I shall emphasize that yeast chromosomal proteins in many ways resemble those of higher eukaryotes. A major difference is the apparent absence of histone Hl in yeast. Nevertheless, yeast chromosomal higher-order structure, and notably its manifestation in chromosome condensation, must have determinants in common with higher eukaryotes, including chromosomal proteins. I shall present examples of these, and some mutants relevant to them. They include DNA topoisomerases, protein kinases and phosphatases, and nuclear matrix (scaffold) proteins. In addition, I would like to stress that nuclear structure may be affected indirectly by proteins that maintain a proper nuclear environment.
Interphase microtubule re-orientation predicts a new cell polarity in wounded pea roots
Authors: Hush Julia M. , Hawes Chris R. , Overall Robyn L.
Published: April 2021

We have developed a useful infiltration and embedding technique for cryosectioning and immunostaining plant tissues, which allows the examination of microtubule (MT) arrays across large areas of sub-epidermal tissue. Using the wound response as a tool for investigating the processes underlying the establishment of a new cell polarity, we have found that a re-orientation of interphase arrays is initiated between 2 and 5 hours post-wounding. This demonstrates that new cell polarity is established sooner after wounding than was previously thought This cytoskeletal shift appears to act as a spatial precursor for the subsequent change in growth axis, which is manifested in the cell expansion planes, and positioning of the pre-prophase band (PPB) and new cell walls. We have demonstrated that interphase MT reorientation precedes and predicts new cell polarity in a higher plant tissue. Possible mechanisms important in orchestrating this dramatic developmental change could be electric fields, generated by ionic wound currents, and the alteration of mechanical stress patterns within the wounded tissue.
Stability of microtubule attachment to metaphase kinetochores in PTK1 cells
Authors: Cassimeris Lynne , Rieder Conlyl. , Rupp Gerald , Salmon E. D.
Published: April 2021

Kinetochore microtubules are known to be differentially stable to a variety of microtubule depolymerization agents compared to the non-kinetochore polar microtubules, but the dynamics of microtubule attachment to the kinetochore is currently controversial. We have examined the stability of kinetochore microtubules in metaphase PtKj spindles at 23 °C when microtubule assembly is abruptly blocked with the drug nocodazole. Metaphase cells were incubated in medium containing 34 pM nocodazole for various times before fixation and processing either for immunofluorescence light microscopy or serial-section electron microscopy. Microtubules not associated with kinetochore fibers disappeared completely in less than 1 min. Kinetochore fibers persisted and shortened, as the spindle poles moved close to the chromosomes over a 10–20 min interval. During this shortening process, the number of kin etochore microtubules decreased slowly. The mean number of kinetochore microtubules was 24±5 in control cells and zero in cells incubated with nocodazole for 20 min. The half-time of microtubule attachment to the kinetochore was ∼7.5 min. These results show that when microtubule assembly is blocked, kinetochore microtubules shorten more slowly and persist about 10 times longer than the labile polar microtubules. If kinetochore microtubules shorten by tubulin dissociation at their plus-ends like the non-kinetochore polar microtubules, then the microtubule surface lattice must be able to translocate through the kinetochore attachment site without frequent detachment occurring.
Effects of thrombospondin antibody on the recovery of endothelial cells from hyperthermia
Authors: Ketis Nika V. , Lawler Jack
Published: April 2021

In addition to the increased synthesis of the classical heat-shock proteins (28000, 71000, 73000, 90000 and 100 000 Mr polypeptides) there is also an increase of thrombospondin in the growth medium of endothelial cells exposed to hyperthermia. The effect of a monoclonal antibody to thrombospondin on the recovery of endothelial cells from hyperthermia as it relates to cytoskeletal organization and cell spreading was assessed. The antibody interacts with the heparin-binding domain of thrombospondin in the extracellular matrix of cells. We report that during recovery from thermal insult at 37 °C, intermediate filaments, stress fibres and micro-tubles show distinct time-recovery characteristics in bovine aortic endothelial cells; that in the presence of this antibody the cytoskeleton is notably altered; that this antibody causes retraction of endothelial cell processes; and that the recovery of the cytoskeleton in endothelial cells exposed to hyperthermia is prevented by the thrombospondin antibody in the time frame examined. Our data suggest that the recovery of cells from heat shock requires the integrity of thrombospondin and its interactions.
Cortical endoplasmic reticulum in plants
Authors: Hepler P. K. , Palevitz B. A. , Lanceele S. A. , McCauley M. M. , Lichtscheidl I.
Published: April 2021

Structural observations provide persuasive evidence for the existence of a cortical network of endoplasmic reticulum (ER) in a large number of plant and animal cells. The network in plants generally possesses a polygonal pattern in which smooth, tubular elements are joined by intervening lamellar segments. The individual elements of ER are often positioned extremely close to the plasma membrane (PM), and may form appositional contacts, but fusion does not occur. The network arises at cytokinesis and establishes continuity between the cortical ER of daughter cells in the form of tightly furled membranetubules that traverse the plasmodesmata. The specific function of the cortical ER complex is unknown but different possibilities seem attractive. It may serve key roles in anchoring the cytoskeleton and in facilitating secretion. The cortical ER might also participate in the communication of signals between the exterior of the cell and cytoplasm. As a consequence of its ability to release and/or sequester Ca, the ER could control the cytoplasmic activity of this ion and thus a host of physiologically and developmentally important reactions.
The composition and morphology of yeast nuclear scaffolds
Authors: Cardenas Maria E. , Laroche Thierry , Gasser Susan M.
Published: April 2021

The yeast nuclear scaffold has been shown to bind with high affinity to genomic sequences that permit autonomous DNA replication of plasmids (ARS elements). We describe here conditions for the isolation of a histone-free nuclear substructure, the nuclear scaffold, from Saccharomyces cerevisiae. We examine the protein composition of this structure, and the conditions under which topoisomerase II, the nuclear factor RAP-1 and RNA polymerase II cofractionate with the scaffold. We find that exposure of nuclei to a combined metal and heat treatment (0.5 mM Cu2+, 37 °C) prior to detergent extraction is required for effective stabilization of these proteins with the scaffold. Electron microscopy of the residual nuclei extracted with non-ionic detergents shows the absence of a typical peripheral lamina structure.
Monoclonal antibody study of the decorated spongiome of contractile vacuole complexes of Paramecium
Authors: Allen Richard D. , Ueno Marilynn S. , Pollard Leonard W. , Fok Agnes K.
Published: April 2021

A monoclonal antibody (mAb) has been developed and selected by immunofluorescence for the radial canals of the contractile vacuole complex (CVC) of Paramecium multimicronucleatum. By applying indirect immunogold labeling to thin frozen sections this mAb has been shown at the electron microscopic level to be specific for the decorated spongiome. We have used the mAb to study the normal interfission appearance as well as developmental stages of the decorated spongiomes. Two decorated spongiomes, presumably involved in water sequestration, radiate as 5-10 bands from unlabeled, circular, 25 μm diameter centers. Two new CVCs arise just anterior to the space occupied by the old spongiomes, the new anterior CVC appearing slightly before the posterior one. Development of the new spongiomes around a 10 gm unlabeled central zone is accompanied by a regression of old spongiome bands until the lengths of these bands in both old and new CVCs are equal just before cell division. After division both old and new spongiome bands grow at equal rates to the same length. Exceptions to the above general scheme, both in number of CVCs in interfission, as well as in position of the new relative to the old CVCs, are also observed.
An analysis of chick limb bud intercellular adhesion underlying the establishment of cartilage aggregates in suspension culture
Authors: Bee James A. , Mark Klaus von der
Published: April 2021

To examine the mechanism of intercellular adhesion in the establishment of limb skeletal elements we have investigated the process of limb bud cell aggregation in vitro. Limb bud cells are aggregation-competent immediately after their trypsin: collagenase dissociation in the absence of calcium. This aggregation is largely Ca2+-independent (CI) and is completely and reversibly inhibited by cycloheximide. In contrast, when limb bud cells are first allowed to recover from Ca2+-free trypsin: collagenase dissociation, aggregation of the surviving population is exclusively Ca2+-dependent (CD) and completely and reversibly inhibited by cycloheximide. The presence of exogenous calcium during initial cell dissociation retains a functional CD aggregation mechanism. However, incubation of such cells with EGTA releases the CD component and converts the cells to a predominantly CI aggregation. Rabbits were immunized with limb bud cells exhibiting the recovered CD aggregation mechanism and the resulting immune sera were screened for their effect on cell aggregation. Relative to pre-immune sera, intact immune IgG agglutinated dissociated limb bud cells whilst immune Fab fragments inhibited their aggregation. The aggregation-inhibiting antiserum recognizes five major limb bud cell surface components with apparent molecular weights of 72K, 50K, 23K, 14.5K and 8.5K (K=103Mr), respectively. Limb bud cell surface plasma membranes were isolated by sucrose gradient density centrifugation and detergent-solubilized proteins coupled to Sepharose 4B with cyanogen bromide. Equivalent cell surface plasma membrane proteins were 125I-iodinated and applied to the affinity column. Limb bud cell surface protein affinity chromatography in the presence of exogenous calcium yields a single protein with an apparent molecular weight of approximately 8.5K. This protein molecule elutes at 0.6 M NaCl, indicating a high affinity, is recognized by the aggregationinhibiting antiserum, and is itself capable of inhibiting CD limb bud cell aggregation. Fab fragments prepared from rabbit antisera specifically directed against the affinity-purified material also inhibit CD limb bud cell aggregation and this inhibition is neutralized by the 8.5K protein. Our data thus demonstrate that CD limb bud cell aggregation is not mediated by fibronectin and/or collagen type I and indicate that this process is governed by a novel 8.5K cell adhesion molecule.
Physiological and structural maturation of a polarised epithelium: The Malpighian tubules of a blood-sucking insect, Rhodnius prolixus
Authors: Skaer H. le B. , Harrison J. B. , Maddrell S. H. P.
Published: April 2021

The development of polarity in a simple epithelium, the Malpighian tubules of Rhodnius, is analysed both ultrastructurally and physiologically. The onset of physiological function, including fluid secretion and the transport of solutes, is determined in late embryos and young hatchling insects and compared with structural development in tubules over a similar period. Two phases of maturation, separated by several days, are detected. The first, during late embryogenesis, involves the development of mature intercellular contacts and the dilation of the lumen, and is associated with the ability to transport specific solutes. The second phase involves the elaboration of the apical and basal membranes and the generation of mitochondria, and is associated with the onset of fluid transport in the tubules and with feeding in 4-day-old hatchlings.
Peroxisomal assembly: Membrane proliferation precedes the induction of the abundant matrix proteins in the methylotrophic yeast Candida boidinii
Authors: Veenhuis Marten , Goodman Joel M.
Published: April 2021

Peroxisomes are massively induced when methylotrophic yeasts are cultured in medium containing methanol. These organelles contain enzymes that catalyze the initial steps of methanol assimilation. In Candida boidinii, a methylotrophic yeast, the peroxisomal matrix (internal compartment) is composed almost exclusively of two proteins, alcohol oxidase and dihydroxyacetone synthase; catalase is present in much lower abundance. Monoclonal and polyclonal antibodies are available against peroxisomal matrix and membrane proteins. These were utilized to correlate the induction of specific proteins with the morphological changes occurring during peroxisomal proliferation. Cells cultured in glucose-containing medium contain two to five small microbodies, which are identifiable by catalase staining and immunoreactivity with a monoclonal antibody against PMP47, an integral peroxisomal membrane protein. Three stages of proliferation can be distinguished when cells are switched to methanol as the carbon source. (1) There is an early stage (within 1 h) in which several peroxisomes develop from a preexisting organelle. This is accompanied by an increase in catalase activity and an induction of PMP47, but no detectable induction of alcohol oxidase or dihydroxyacetone synthase is observed. (2) From 1 to 2.5 h there is further division of these microbodies until up to 30 small peroxisomes generally are present in each of one or two clusters per cell. Induction of alcohol oxidase, dihydroxyacetone synthase and PMP20, a protein that is distributed in the matrix and membrane, is detectable during this time. Serial sections reveal that some peroxisomes remain uninduced while others undergo proliferation. Such sections also show no obvious connections between peroxisomes within clusters. (3) After 2.5 h there is a decrease in the number of peroxisomes per cell (caused at least in part by the movement of organelles into buds) but an increase in volume per peroxisome, until a steady state is reached by 5–10 h.
Cardiac myocyte gap junctions: evidence for a major connexon protein with an apparent relative molecular mass of 70,000
Authors: Harfst E. , Severs N.J. , Green C.R.
Published: April 2021

It is widely accepted that there is a family of gap junction connexon proteins, their distribution appearing to vary with tissue type and species. In cardiac tissues the major junctional channel component identified is a 43K (K = 10(3) Mr) polypeptide. Using a gap junction isolation protocol in which low temperatures are maintained, and which is detergent-free, we have identified a second gap junction-related protein in cardiac tissues with an apparent relative molecular mass of 70,000. Antibodies raised to three synthetic peptides matching portions of the 43K gap junction protein cDNA sequence cross-react with the 70K protein, but biochemical studies indicate that these proteins are distinct from one another. The structures that contain the 70K protein are susceptible to fragmentation at warm temperatures, and by electron microscopy this can be correlated with loss of ‘minidomains’ within the junctional plaque. Using a gap junction enriched-fraction prepared from purified isolated adult myocytes we show that both the 43K and 70K gap junction proteins are present in ventricular cardiac myocytes. In such preparations, and those from whole heart, the 70K protein appears to be the major gap junction-related protein present.
Domain-specific changes of ciliary striated rootlets during the cell cycle in the hypotrich ciliate Paraurostyla weissei
Authors: Jerka-Dziadosz Maria
Published: April 2021

The dynamics of striated ciliary rootlets (cr) during development of ciliary structures in cells of the hypotrich ciliate Paraurostyla weissei was studied by immunostaining with polyclonal antibody raised against isolated cr of Paramecium tetraurelia. Wildtype cells and two mutants: mlm showing multiple left marginal cirri and mlm/pl showing in addition a certain degree of pattern lability were used to study the boundaries of particular cortex domains.

In interphase cells, cr are attached to all left marginal and caudal cirri and to only the posterior third of the right marginal cirri, cr appear in all nonoral primordia during the first wave of basal body proliferation. After nucleation and early elongation of cr, some cr undergo site-specific regression or stabilization. The spatial deployment of these different modes of development corresponds to specific cortical domains.

In mlm/pl mutants, where specific cortical domains are broadened, changes in the cr system are characteristic for a given domain, regardless of its broadening, but boundaries between adjacent domains are weakened.
Pharmacological evidence for a role for cyclic AMP signalling in Dictyostelium discoideum slug behaviour
Authors: Darcy P. K. , Fisher P. R.
Published: April 2021

Phototaxis and thermotaxis by Dictyostelium discoideum slugs on water agar were impaired by the presence in the agar of adenosine, which is a cyclic AMP receptor antagonist in aggregating amoebae. Caffeine, and presumably its analogue theophylline, inhibit cyclic AMP signalling in aggregating amoebae of D. discoideum. Both compounds perturbed slug behaviour in a similar manner to adenosine, as did both ammonium and sulphate ions. (NH4)3SO4 is known to perturb cyclic AMP binding to its receptor, and ammonia is an inhibitor of cyclic AMP signalling in aggregating amoebae. The receptor agonist, cyclic AMPS, disrupted slug organization and impaired phototaxis when present at concentrations high enough to saturate cyclic AMP receptors and compete effectively with endogenous cyclic AMP signals of similar magnitude to those observed during aggregation. Taken together with the considerable circumstantial evidence for cyclic AMP signalling in slugs, these results support a role for cyclic AMP signalling in slug behaviour.
Cell cycle regulation of p34 cdc2 kinase activity in Physarum polycephalum
Authors: Ducommun Bernard , Tollon Yvette , Garés Michéle , Beach David , Wright Michel
Published: April 2021

The regulation of the mitotic histone H1 kinase activity has been analyzed during the naturally synchronous cell cycle of Physarum polycephalum plasmodia. The universal binding property of the pl3suo1Schizosaccharomyces pombe gene product was used to precipitate and assay the cdc2 histone H1 kinase activity. The kinase activity peaks at the beginning of metaphase and its decline, which requires protein synthesis, appears to be an early event during the metaphase process. Microtubular poisons, temperature shifts and DNA synthesis inhibitors were used to perturb cell cycle regulatory pathways and characterize their effects on cdc2 kinase activation. Our results suggest that the full activation of the mitotic kinase requires at least two successive triggering signals involving microtubular components and DNA synthesis.
Promotion of transendothelial neutrophil passage by human thrombin
Authors: Moser René , Groscurth Peter , Fehr Jorg
Published: April 2021

Thrombin has been reported to elicit two temporally different effects on neutrophil-endothelial interaction, categorically described as ‘neutrophil adhesion’: one expressed within a few minutes, the other after several hours of endothelial preincubation. Prolonged activation resulted in often elongated, tightly interacting neutrophils in contact with human umbilical vein endothelial cells (HUVE) mainly at the intercellular region. In contrast, the neutrophil-endothelial interaction due to short-time priming with thrombin was dominated by round, randomly distributed neutrophils, loosely adhering on the endothelial surface. Sheer stress, introduced to characterize these morphological entities in a quantitative manner, clearly defined the neutrophil response due to short time priming as sheer stresslabile compared to the sheer stress-resistant interaction expressed after prolonged preincubation of HUVE with thrombin. Quantitative determination of neutrophil transendothelial migration served to demonstrate the relevance of such differences in neutrophil binding with respect to subsequent layer penetration. Dependent upon endothelial protein synthesis and inhibited by antithrombin-III-heparin and hirudin, neutrophil layer penetration only occurred after prolonged activation of HUVE whereas the neutrophil-endothelial interaction due to short time priming with thrombin was limited to superficial neutrophil adhesion.

We conclude that thrombin can be added to the list of activators capable of inducing the neutrophil passage-guiding principle, an effect that has recently been detected as closely related to neutrophil adhesion to endothelial ligands expressed upon activation with interleukin-1 and tumor necrosis factor. This biological activity, which can be separated from superficial attachment of polymorphonuclear leukocytes (PMN) to endothelial cells, is a catalytic sitedependent and late phase-specific effect of thrombin on HUVE.
Microgravity decreases C-fos induction and serum response element activity
Authors: groot Rolf P. de , Rijken Philip J. , Hertog Jeroen Den , Boonstra Johannes , Verkleij Arie J. , Laat Siegfried W. De , Kruijer Wiebe
Published: April 2021

Several studies have shown that altered gravity conditions influence mammalian cell growth and differentiation. The molecular mechanisms underlying these effects, however, remain relatively obscure. In this paper we show that microgravity reached in a sounding rocket strongly decreases epidermal growth factor (EGF)-induced expression of the protooncogenes c-fos and c-jun, which are both implicated in the regulation of proliferation and differentiation. Decreased activity of the serum response element (SRE), present in the c-fos promoter-enhancer region, is probably responsible for the decrease in EGF-induced c-fos expression. In addition, we show that gravity alterations differentially modulate distinctive signal transduction pathways, indicating that gravity-dependent modulations of mammalian cell proliferation are unlikely to be caused by a nonspecific stress response of the cell.
Synthetic epidermal pentapeptide and related growth regulatory peptides inhibit proliferation and enhance differentiation in primary and regenerating cultures of human epidermal kératinocytes
Authors: Jensen Peter K. A. , Elgjo Kjell , Laerum Ole D. , Bolund Lars
Published: April 2021

A pentapeptide that inhibits proliferation of mouse epidermal keratinocytes in vivo and in vitro has been purified from mouse skin extracts. In the present study the effect of a synthetic analog of the epidermal pentapeptide on proliferation and differentiation of cultured human epidermal kératinocytes was investigated. In young, rapidly growing primary cultures the pentapeptide caused a dramatic decrease in mitotic activity and also induced pronounced changes in the balance between kinetically defined subpopulations of proliferating cells. A dipeptide derived from the pentapeptide was found to be at least as potent A serine derivative of a hemoregula-tory peptide also seemed to be active. When tested in epidermal cultures regenerating after removal of the suprabasal cell layers, both the pentapeptide and the dipeptide were shown to cause a delay in the proliferative response. Both peptides were also able to stimulate early (increase in cell size) and late (cornified envelope formation) events in the differentiation pathway of the kératinocyte. The apparent stimulatory effect on differentiation was most clearly seen in regenerating cultures, whereas the effect on primary cultures varied with the experimental set-up. It is suggested that homologous epidermal peptide(s) may play a major role in the regulation of human epidermal homeostasis.
Desmosomal glycoproteins 2 and 3 (desmocollins) show N-terminal similarity to calcium-dependent cell-cell adhesion molecules
Authors: Holton J. L. , Kenny T. P. , Legan P. K. , Collins J. E. , Keen J. N. , Sharma R. , Garrod D. R.
Published: April 2021

The N-terminal sequence of a mixture of desmosomal glycoproteins 2 and 3 (dg2/3, desmocollins) from bovine nasal epidermis, prepared by electro-elution from polyacrylamide gels, was determined by solidphase Edman degradation. A sequence of 23 amino acids was obtained. This showed 43% identity with that of the N terminus of the calcium-dependent cell adhesion molecule, N-cadherin. A lesser degree of identity with other members of the cadherin–uvo– morulin-L-CAM family was also found. In order to confirm that the sequence was derived from the dg2/3 molecules a rabbit antiserum was raised against a synthetic peptide corresponding to the sequence, conjugated to keyhole limpet haemocyanin (KLH). The antiserum obtained showed high (titre) activity against both the peptide and KLH in ELISA. Each activity could be specifically adsorbed with the appropriate ligand. The antiserum reacted specifically with both dg2 and dg3 of bovine nasal epidermis on immunoblots, this binding was blocked by the N-terminal peptide but was unaffected by KLH. The identity of dg2 and -3 in these preparations was confirmed by immunoblotting with two monoclonal antibodies and one polyclonal antiserum raised against the whole molecules. The N-terminal peptide antiserum was shown to bind to the intercellular space of desmosome profiles by immunoelectron microscopy on ultra-thin frozen sections. One of the two monoclonal antibodies (07–4D) also reacted with the desmosomal intercellular space. dg2 and -3 were shown by Staphylococcus aureus V8 protease digestion to have identical one-dimensional peptide maps. Both the N-terminal antiserum and 07–4D reacted with a V8 fragment of 19000Mr derived from dg2 and dg3. The other monoclonal antibody (52–3D), which has previously been shown to react with the cytoplasmic regions of dg2/3, recognised V8 fragments of lower molecular weight. These results indicate that the dg2/3 molecules of epidermal desmosomes are related to a family of calcium-dependent adhesion molecules, demonstrate that the N-termini of the molecules are located extracellularly, and support the view that they are directly implicated in desmosomal adhesion.
Differential effects of myosin-antibody complexes on contractile rings and circumferential belts in epitheloid cells
Authors: Zurek Barbara , Sanger Jean M. , Sanger Joseph W. , Jockusch Brigitte M.
Published: April 2021

The role of myosin filaments during assembly and activity of microfilament rings was analyzed by microinjecting epitheloid cells (PtK2 and LLC-PK1 kidney cell lines) with specific anti-myosins. Six monoclonal antibodies directed against the light meromyosin (LMM) region of the myosin molecule were characterized with respect to epitope location, and their effects on actin-activated MgATPase as well as on assembly, structural integrity and stability of myosin filaments. All of these antibodies recognized LLC-PK1 myosin, but only three reacted with PtK2 myosin. The remaining three served as matching controls in experiments with this cell line. When injected in amounts sufficient to yield an excess of antibody over myosin, the reactive antibodies significantly delayed formation and constriction of the contractile ring in mitotic cells. These rings contained less myosin, but not less actin, than the controls. This indicates that the recruitment and alignment of actin in the cleavage furrow can occur independently of other components of the contractile ring.

After completion of cytokinesis, the majority of the injected cells was unable to assemble a normal circumferential belt. This resulted in defective epitheloid sheets. Approximately one third of these cells showed grossly distorted cell shapes and an increase in locomotory activity.

All these changes were fully reversible with time, suggesting that the effects of the antibodies were overcome by protein synthesis. The differential sensitivity seen between contractile rings and peripheral belts is discussed with respect to differences in their architecture, stability and proposed function.
Microinjection of covalently cross-linked actin oligomers causes disruption of existing actin filament architecture in PtK2 cells
Authors: Handel S. E. , Hendry K. A. K. , Sheterline P.
Published: April 2021

Experiments were performed to determine the effects of interrupting the flux of actin monomers between unpolymerised and polymerised pools in PtK2 cells by (1) microinjecting exogenous polymerisation nuclei and (2) blocking endogenous assembly sites with low concentrations of cytochalasin D.

Fluorescent actin oligomers were prepared by glutaraldehyde cross-linking F-actin derivatised at cysteine-374 with 5-iodoacetamido-fluorescein. These oligomers caused rapid nucleation of polymerisation of pyrene-labelled actin in vitro. Different numbers of polymerisation nuclei were injected into PtK2 cells and the cells were incubated for various times. Microinjection of between 1.2×104 and 1.8×104 nuclei per cell resulted in the complete disassembly of existing actin filament structures in nearly half of the cells within 15 min. Existing structures in such cells were replaced by foci of polymerised actin, which colocalised with concentrations of nuclei. Injection of increasing numbers of nuclei between 3×103 and 1.2 ×104 caused fragmentation of stress fibres in an increasing proportion of cells, whereas injection of less than 3× 103 caused no obvious effects even over a 90 min incubation period. These data indicate that the degree of disruption of stress fibres was a function of the number of nuclei injected, but that it was less dependent on the incubation time. The minimum number of injected nuclei causing complete disruption of actin filament structures provides an estimate for the number of endogenous nuclei involved in filament turnover, whereas the minimum period for reorganisation (about 15 min) implies a maximum time for the complete turnover of actin in the system.

Treatment of cells with low concentrations (3× 10−8 to 10−6m) of cytochalasin D, which prevents assembly at barbed-end assembly sites, also caused destruction of existing filament structures.

These data indicate that either substitution of endogenous assembly sites by exogenous nuclei, or blockage of existing endogenous assembly sites by cytochalasin, subverts the cycle of assembly and disassembly of normal actin filament architectures in PtK2 cells, leading to the assembly of aberrant structures.
Phenotypic modulation in sub-populations of human articular chondrocytes in vitro
Authors: Archer Charles W. , McDOWELL Jenny , Bayliss Michael T. , Stephens Myra D. , Bentley George
Published: April 2021

Human articular cartilage has been separated into surface (approx. 15% of tissue depth) and deep zones (remaining tissue) and the constituent chondrocytes released by enzymic digestion. Subsequent culture either as a low density monolayer or as a suspension over agarose revealed distinct morphological and synthetic behaviour in the two populations. Whilst in monolayer these morphological differences disappeared with time in culture, over agarose they remained. Surface zone cells formed two types of cell cluster; one that was highly cellular with little extracellular matrix, and the other less frequent, which formed copious amounts of fibrillar matrix. Both types of cluster were surrounded by a layer of flattened chondrocytes. In contrast, deep cells formed a single cluster type that lacked a surrounding cell layer, but formed large amounts of sparse cartilage-like matrix and comprised morphologically typical chondrocytes. In monolayer, both populations gradually ceased to synthesise cartilage matrix components with the exception of link protein. In suspension, whilst the chondrogenic phenotype per se was preserved, there was, nevertheless, a loss in qualitative synthetic heterogeneity, which exists between surface and deep cells, that was not accompanied by changes in the differential rate of 35S incorporation into proteoglycan. Under these conditions, surface cells that normally do not synthesise keratan sulphate initiated de novo synthesis of this glycosaminoglycan. Consequently, it appears that the observed modulation in synthetic ability of the cell sub-populations is independent of the cluster morphology, which, once established, remains constant throughout the culture period.
The pattern of metalloproteinase expression by corneal fibroblasts is altered by passage in cell culture
Authors: Fini M. Elizabeth , Girard Marie T.
Published: April 2021

We have examined the pattern of expression of four different matrix metalloproteinases (MMPs), collagenase, stromelysin, 92 kD gelatinase, and 72 kD gelatinase, by primary and passaged cultures of rabbit corneal fibroblasts. Primary cultures of this cell type have previously been shown to reproduce the normal tissue regulation of collagenase expression. We demonstrate qualitative and quantitative changes in the pattern of MMP expression as the cells are passaged in culture. Only a single MMP, 72 kD gelatinase, is constitutively expressed by primary fibroblast cultures. Phorbol myristate acetate (PMA) treatment upregulates expression of 72 kD gelatinase and turns on the expression of collagenase and stromelysin, as well as 92 kD gelatinase. However, the degree to which MMP expression is induced is minimal. Cells subcultured but a single time constitutively produce not only 72 kD gelatinase, but also collagenase and stromelysin. In addition, PMA treatment upregulates expression of collagenase, stromelysin and 92 kD gelatinase to high levels. In contrast, the expression of 72 kD gelatinase is repressed by treatment of passaged cell cultures with PMA. Our data indicate that the cell does not simply turn the MMP genes on or off, as a group, in response to various agents, but that it has the capacity for fine control over which MMPs are expressed and the degree to which each is expressed. Changes in MMP protein expression induced by PMA treatment are correlated with changes in specific mRNA levels in passaged cultures. The kinetics of mRNA accumulation suggest that the MMP genes can respond to multiple intracellular signals initiated in a temporal cascade by PMA. It is the combined effects of the individual signals on the accumulation of specific mRNAs that must determine the ultimate pattern of MMP protein expression. The distinct patterns of MMP expression produced by primary and passaged cell cultures may be analogous to patterns of expression that might occur under particular in vivo conditions.
Convergence of apical and basolateral endocytic pathways at apical late endosomes in absorptive cells of suckling rat ileum in vivo
Authors: Fujita Mamoru , Reinhart Frederick , Neutra Marian
Published: April 2021

Absorptive cells of the intestinal epithelium endocy-tose proteins from both apical and basolateral membrane domains. In absorptive cells of suckling rat ileum, luminal protein tracers first enter an apical tubulovesicular endosomal system, then enter larger apical endosomal vesicles and multivesicular bodies (MVB), and finally are delivered to a giant supranuclear lysosomal vacuole. To determine whether proteins endocytosed from the basolateral domain in vivo enter the same endosomal or lysosomal compartments as those taken up from the apical side, we simultaneously applied cationized ferritin (CF) apically (by intra-luminal injection) and horseradish peroxidase (HRP) basally (by intravenous injection), and examined absorptive cells after 3 min to 60 min using light, electron and fluorescence microscopy. At early times, CF and HRP entered separate endosomal compartments at apical and basolateral poles. At no time did HRP enter the apical tubulovesicular system, and CF never entered early basolateral endosomes. After 15 min, however, both tracers appeared together in large late endosomes and MVB located apically, above the giant vacuole. From 15 to 60 min both tracers accumulated in the giant vacuole. Membranes of some apical late endosomes, all apical MVB, the giant vacuole, and occasional sub-nuclear vesicles contained immunoreactive lgpl20, a glycoprotein specific to late compartments of the endosome–lysosome system. These results show that highly polarized intestinal epithelial cells have separate apical and basolateral early endosomal compartments, presumably to maintain distinct membrane domains while allowing endocytosis and recycling of membrane from both surfaces. Apical and basolateral endocytic pathways, and presumably vesicles delivering hydrolytic enzymes and lysosomal membrane components, converge at the apical late endosome.
Ehrlich ascites tumour cells show tissue-specific adherence and modify their shape upon contact with embryonic fibroblasts and myotubes
Authors: Wójciak Beata , Korohoda Włodzimierz
Published: April 2021

Adhesiveness of Ehrlich ascites tumour (EAT) cells to glass, to mouse peritoneal membrane, living and aldehyde-fixed mouse embryo fibroblasts and chick embryo fibroblasts, myoblasts and myotubes was investigated. The ascitic EAT cells (and leukaemia L1210 cells) did not adhere to glass and peritoneum but readily adhered to embryo fibroblasts, myoblasts and myotubes. The attachment was followed by cell spreading and migration. Fixation of fibroblasts or myogenic cells with aldehydes did not prevent ascitic cells from attaching but reduced the rate of spreading. Only direct interaction of ascitic cells with embryo myoblasts or fibroblasts induced changes in tumour cell adhesiveness followed by cell spreading and locomotion. These results are discussed in relation to an observation that ascitic cells growing as a cell suspension intraperitoneally grow as a solid tumour when injected subcutaneously.
Calcium depletion of Dictyostelium cells selectively inhibits cyclic nucleotide phosphodiesterase synthesis at a post-transcriptional step
Authors: Coukell M. B. , Cameron A. M.
Published: April 2021

We reported previously that Ca2+ deprivation of Dictyostelium discoideum amoebae inhibits both the induction by cyclic AMP of soluble cyclic nucleotide phosphodiesterase (PD) activity in wild-type cells and the constitutive production of this activity in mutant HC35 cells. In this study, the mutant strain has been used to characterize this phenomenon further and to attempt to identify the Ca2+-dependent step(s) in PD production. Incubation of HC35 cells with 0.9 mM EGTA increased the rate of 4SCa2+ efflux from preloaded cells and partially inhibited PD production. The effect of EGTA on both processes was enhanced considerably by the presence of 1 fiM A23187. This concentration of ionophore alone had little effect on either Ca2+ efflux or PD production. Addition of 1 pM free extracellular Ca2+ to amoebae incubated with EGTA and A23187 increased PD production two-to sixfold. Ca2+ depletion did not appreciably inhibit total protein synthesis or total protein glycosylation as determined by the incorporation of [3H]leucine or [3H]mannose, respectively. Also, the abundance of PD mRNAs was approximately the same in Ca2+-depleted and control cells. In contrast, immunoblot analysis of extracellular fluids and intracellular extracts of these cells revealed a dramatic reduction (up to 85%) in the levels of PD-related polypeptides associated with the Ca2+-depleted cells. These results suggest that Ca2+ deprivation selectively inhibits PD production by impairing a post-transcriptional process.
Computer modelling of Tetrahymena axonemes at macromolecular resolution: Interpretation of electron micrographs
Authors: Sugrue P. , Avolio J. , Satir P. , Holwill M. E. J.
Published: April 2021

A computer-generated model of the structural arrangement of the complete 9+2 ciliary axoneme of Tetrahymena at macromolecular resolution (4 nm) is presented. The model reconciles detailed information about subcomponents from negative-stained, thin-section and freeze-fracture electron micrographs, integrating the images into a consistent three-dimensional picture. This illuminates problems such as the requirement for compaction of dynein to form the arm, difficulties in visualization of the circumferential links, construction of the central sheath, and the comparative periodicities of the inner and outer arms. The model is pragmatic in that it is flexible and easily changed, as new information becomes available. It is also useful in the development of dynamic concepts, such as a spatial description of the dynein cross-bridge cycle, which is illustrated, or relationships between adjacent doublets during sliding and bending.