bluuebunny/crossref_metadata_2025 huggingface dataset:
The complex cell cycle of the dinoflagellate protoctistCrypthecodinium cohniias studiedin vivoand by cytofluorimetry
Authors: Bhaud Yvonne , Salmon Jean-Marie , Soyer-Gobillard Marie-Odile
Published: April 2021

The complete cell cycle of the dinoflagellate Crypthecodinium cohniiBiecheler 1938 was observed in vivo in a synchronized heterogeneous population, after DAPI staining of DNA. In a given population, the relative nuclear DNA content in each class of cell was measured using a new numerical image-analysis method that takes into account the total fluorescence intensity (FI), area (A) and shape factor (SF). The visible degree of synchronization of the population was determined from the number of cells with a nuclear content of IC DNA at ‘synchronization’, time 0. One method of synchronization (method 1), based on the adhesiveness of the cysts, gave no better than 50% synchronization of the population; method 2, based on swimming cells released from cysts cultured on solid medium, gave 73% of cells with the same nuclear DNA content. A scatter plot of data for FI versus A in the first few hours after time 0 showed that the actual degree of synchronization of the population was lower.

The length of the C. cohnii cell cycle determined in vivo by light microscopy was 10, 16 or 24 h for vegetative cells giving two, four or eight daughter cells, respectively. Histograms based on the FI measurements showed that in an initially synchronized population observed for 20 h, the times for the first cell cycle were: G1 phase, 6 h; S phase, 1 h 30 min; G2+M, lh 30 min, with the release of vegetative cells occurring 1 or 2h after the end of cytokinesis. The times for the second cell cycle were G1+S, 3h; G2+M, 2h.

FI and A taken together, suggested that the S phase is clearly restricted, as in higher eukaryotes. A and SF, taken together, showed that the large nuclear areas were always in cysts with two or four daughter cells. FI and SF, taken together, showed that the second S phase always occurred after completion of the first nuclear division.

Our data concerning the course of the cell cycle in C. cohnii are compared with those from earlier studies, and the control of the number of daughter cells is discussed; this does not depend on the ploidy of the mother cell.
The membrane skeleton of Pseudomicrothorax: II. biochemical and immunological characterization of the glycosylated cytoskeletal proteins
Authors: Huttenlauch Irm , Peck Robert K.
Published: April 2021

The membrane skeleton, or epiplasm, is part of the structurally complex ciliate cortex. It is thought to have skeletal functions concerning the spatial organization of cortical elements such as the basal bodies.

Here we report the biochemical and immunological characterization of some components of the purified epiplasm of Pseudomicrothorax dubius. The epiplasm proteins consist of two quantitatively major groups of proteins, one of 76-80 ×103Mr, the other of ll-13×103Mr, which appear to be the principal structural elements of the epiplasm, and a series of minor components of 62-18×103Mr. Based upon lectin labeling and glycosidase treatment, some of the latter have been identified as glycoproteins. Using affinity-purified antibodies specific for individual glycoproteins or groups of glycoproteins, we were able to localize them in situ by immunoelectron microscopical methods. This in situ localization demonstrates that the glycosylated epitopes, unlike the glycoresidues of membrane proteins, are distributed throughout the entire epiplasmic layer rather than being restricted to regions adjacent to the cortical membranes. Thus, these proteins represent glycosylated, cytoskeletal elements. At least one of these glycoproteins (Mr 62×103) shows positive immunoreactivity with a monoclonal antibody (Pruss anti-IFA) recognizing most intermediate filament (IF) proteins, indicating that IF proteins might be present in protozoan cytoskeletons.
Distribution of membranes and the cytoskeleton during cell plate formation in pollen mother cells of Tradescantia
Authors: Schopfer Christel R. , Hepler Peter K.
Published: April 2021

The cellular pattern and distribution of membranes have been analyzed during cytokinesis in pollen mother cells of Tradescantia and compared with those of actin microfilaments (MFs) and microtubules (MTs). Membranes have been stained with DiOC6(3) and MFs with rhodamine-labeled phal-loidin (RP); analysis has been carried out on the confocal laser scanning microscope. MTs have been visualized as birefringent elements in the polarized light microscope. The results show that when the interzone first appears in mid anaphase it contains an even distribution of membranes. However, by late anaphase these elements have been cleared away, leaving the interzone largely devoid of DiOC6(3)-positive material. MTs are found throughout this zone, while MFs appear in two non-overlapping sets on both sides of the cell equator. Thereafter membrane elements reappear in the interzone, but only along the equatorial line of the forming cell plate. Presumably these equatorial elements are composed of endoplasmic reticulum and Golgi vesicles, since the larger organelles, including amyloplasts and mitochondria, are excluded from the phragmoplast. MFs, like MTs, arrange preferentially normal to the cell plate, forming a dense array on both sides, but being absent from the zone occupied by the membranes. By contrast, the parallel set of MTs, while excluding larger organelles from the phragmoplast, intermingle with the membrane elements in the cell equator. As cytokinesis proceeds membranes continue to concentrate on the cell plate as indicated by its marked increase in staining with DiOC6(3). From a consideration of spatial and temporal organization of the phragmoplast components it is reasonable to suggest that both cytoskeletal components participate in the aggregation of vesicles that give rise to the cell plate. Membranes, on the other hand, through the provision of surface binding sites and/or through the regulation of the cytoplasmic calcium ion concentration, might be involved in the assembly and stabilization of the cytoskeleton.
Distribution of PCNA in Drosophila embryo during nuclear division cycles
Authors: Yamaguchi Masamitsu , Date Takayasu , Matsukage Akio
Published: April 2021

An immunocytochemical method using a specific antibody was employed to detect the proliferating cell nuclear antigen (PCNA) in Drosophila embryos during the first 13 nuclear division cycles. Strong nuclear staining with the anti-PCNA antibody was observed at interphase throughout 13 cycles. Metaphase chromosomes were not stained throughout these cycles. The chromosomal (nuclear) staining reappeared at anaphase until cycle 10 and at telophase in cycle 11. During cycles 12 and 13, nuclear staining was detected exclusively at interphase. Relatively uniform staining of syncytial cytoplasm was observed throughout mitotic phases until cycle 9. In the following cycles, strong staining in both the central yolk mass and the cortical layer of cytoplasm was detected at metaphase and telophase. During interphase of cycles later than the 9th, staining in the central yolk mass got much fainter and that in the cortical cytoplasm completely disappeared. These results suggest that the PCNA dissociates from chromosomes at metaphase; then in later mitotic phases, it is transported from the syncytial cytoplasm into nuclei to participate in formation of the active DNA-replication enzyme complexes.
Calcium sequestration in the golgi apparatus of cultured mammalian cells revealed by laser scanning confocal microscopy and ion microscopy
Authors: Chandra Subhash , Kable Eleanor P. W. , Morrison George H. , Webb Watt W.
Published: April 2021

Co-localization of the elements calcium, potassium, sodium and magnesium with sequestering organelles has been achieved by application of two microscopy techniques on the same cell. Organelles were first localized by laser scanning confocal microscopy (LSCFM) using fluorescent organelle stains. The same cells were then analyzed for elemental distribution with ion microscopy. This approach has identified a perinuclear region of prominent total calcium concentration with the Golgi apparatus. Live cells were fluorescently stained with C6-NBD-cera-mide for labeling the Golgi apparatus prior to cryogenic preparation and freeze-drying, and imaged with LSCFM for Golgi localization; identical cells were then analyzed with ion microscopy to image subcellular distributions of total calcium, potassium, sodium and magnesium. In three cell lines, LLC-PKi porcine kidney epithelial cells, Swiss 3T3 mouse fibroblast cells and L5 rat myoblast cells, the Golgi regions contained significantly higher total calcium concentrations than any other region of the cell (as measured at the spatial resolution of ion microscopy of about 0.5 /an). Intracellular potassium, sodium and magnesium were homogeneously distributed throughout the cell and did not show this pattern. Measurements of depletion of calcium by exposure to calcium-free medium showed that the Golgi apparatus was substantially more resistant to calcium depletion than all other regions of these cells, but sequestered Ca2+ could be released from the Golgi by exposing the cells to calcium ionophore A23187. The Golgi apparatus appears to sequester about 5% of the total cell calcium in LLC-PKi cells, about 2.5% in 3T3 cells and L5 cells.
The effect of permeant and impermeant osmoticants on exocytosis in guinea pig sperm
Authors: Green D. P. L.
Published: April 2021

Guinea pig sperm were suspended in calcium-containing medium supplemented with various concentrations of the tetrasaccharide, stachyose. At concentrations up to and including 0.6 M, stachyose was without effect on the A23187-induced acrosome reaction. At 1.0 M stachyose, >97% of sperm retained their acrosome after exposure to A23187, as judged by light microscopy. Electron microscopy demonstrated, however, that exocytotic membrane fusion had occurred, although with substantial retention of the acrosomal matrix. Sperm incubated in 1.0 M stachyose solutions also underwent exocytotic membrane fusion in the absence of A23187 and external calcium.

Sperm suspended in 0.175 M ammonium chloride solution progressively lost motility over 30 min, but without acrosomal swelling. By contrast, sperm in 0.19 M ammonium acetate underwent substantial swelling of the acrosome within 2-5 min. 70-80% of these sperm were able to exclude the vital dye propidium iodide with their acrosomes swollen. These sperm underwent acrosomal shrinkage if resuspended in normal medium within 5-10 min, and the majority (60-70%) recovered some motility. These sperm could undergo an A23187-induced acrosome reaction. Electron microscopy indicated that swelling in ammonium acetate solution solubilizes much of the acrosomal matrix and causes internal fusion between adjacent regions of the outer acrosomal membrane. There was no exocytotic membrane fusion in ammonium acetate solution, however.

The evidence suggests that there is no stachyose osmolality for guinea pig sperm which will suppress the membrane fusion associated with exocytosis, and that sufficiently high osmolalities cause exocytotic membrane fusion in the absence of calcium. The evidence also suggests that release of the normal osmotic compression of the acrosomal matrix does not cause local blebbing of the acrosomal membrane, nor does it render the membrane fusigenic. The data are consistent with the model for exocytosis in which no granule swelling occurs prior to membrane fusion.
Assembly of actin-containing cortex occurs at distal regions of growing neurites in pc12 cells
Authors: Sanders Mitchell C. , Wang Yu-Li
Published: April 2021

Although actin filaments are known to be localized in the cortex of axons and in the growth cones of nerve cells, it is unclear how actin-containing structures are assembled during nerve growth. We have studied the formation of actin structures in growing neurites by microinjecting fluorescent phalloidin or actin into PC 12 neuron-like cells to label endogenous actin filaments. Upon stimulation of neurite growth in cells microinjected with fluorescent phalloidin, little or no fluorescence was detected in nascent growth cones and adjacent neurites despite the presence of actin filaments in these regions, suggesting that actin filaments were primarily formed by de novo assembly rather than the transport and reorganization of pre-existing, phalloidin-labeled actin filaments. Time-lapse observations of the distribution of phalloidin-labeled actin filaments during neurite elongation confirmed that fluorescence associated with pre-existing neurite cortex spread out more slowly than the elongation of neurites. Furthermore, when a dark spot was photobleached with a laser microbeam along neurites of cells microinjected with either fluorescent phalloidin or actin, the spot showed no appreciable translocation during active neurite elongation. Taken together, these results suggest that de novo assembly of actin filaments plays a crucial role in the formation of growth cones and adjacent cortex in the distal region of neurites, but does not appear to require the anterograde or retrograde transport of cortical filaments, or the passive stretching of the proximal segment of the neurite cortex.
Laminin and basement membrane-associated microfilaments in wild-type and mutant Drosophila ovarian follicles
Authors: Gutzeit Herwig O. , Eberhardt Wolfgang , Gratwohl Eugen
Published: April 2021

The localization of the extracellular matrix glycoprotein laminin was studied using polyclonal antilaminin antibodies. The laminin patterns of the basement membranes of the muscular epithelial sheath that envelops the ovariole were conspicuously different from those of the basement membrane of the follicular epithelium. In the latter structure laminin was stained in a pattern of parallel stripes oriented perpendicular to the long axis of the follicle; microfilament bundles at the adjacent basal side of the follicle cells have the same orientation. At late vitellogenic stages the orientation of the microfilaments remained the same while the laminin stripes were no longer visible. The orientation of laminin and F-actin was abnormal in follicles of the egg-shape mutant kugel, which produces shorter and thicker eggs than wild-type flies. This phenotype might result from the disturbance of the normal circular microfilament and/or laminin pattsern.
A 33 kda protein with sequence homology to the ‘laminin binding protein’ is associated with the cytoskeleton in hydra and in mammalian cells
Authors: Keppel E. , Schaller H. C.
Published: April 2021

In hydra and in mammalian cells the monoclonal antibody V recognises an epitope which colocalises with cytoskeletal structures. Using this antibody for expression screening, a cDNA clone (955 bp) was isolated from hydra, which covers an open reading frame for a protein of 294 amino acids with a calculated molecular mass of 32.8 kDa. Northern blot analysis of hydra RNA resulted in a single mRNA species of 1.2 kb, and primer extension experiments proved this to be the full length message. 218 residues at the amino terminus of the hydra protein show extensive homology (73.5%) to a human protein designated ‘laminin binding protein’. The carboxyl-terminal 76 amino acids possess no significant similarity (20%). The monoclonal antibody V, which recognises an epitope in this carboxyl-terminal part, reacts in Western blots, both in hydra and in mammalian cells, with a protein of 33 kDa and not with the 45 kDa ‘laminin binding protein’. The 33 kDa protein is not extracellular or transmembrane, but has a strictly intracellular location as indicated by its amino acid sequence and by immunocytochemical and cell fractionation studies.

In non-dividing mammalian cells the 33 kDa protein colocalises with filamentous structures; in dividing cells it dissociates from it and concentrates centrally. Presence of the SPLR-sequence, which is the consensus phosphorylation motif for the p34cdc2 kinase, links this 33 kDa protein to events occurring during the cell cycle.
Abberant chemotaxis and differentiation in Dictyostelium mutant fgdc with a defective regulation of receptor-stimulated phosphoinositidase c
Authors: Bominaar Anthony A. , Kesbeke Fanja , Snaar-Jagalska B. Ewa , Peters Dorien J.M. , Schaap Pauline , Haastert Peter J. M. Van
Published: April 2021

Dictyostelium cells use extracellular cyclic AMP both as a chemoattractant and as a morphogen inducing cell-type-specific gene expression. Cyclic AMP binds to surface receptors, activates one or more G-proteins, and stimulates adenylate cyclase, guanylate cyclase and phosphoinositidase C. Mutant fgdC showed aberrant chemotaxis, and was devoid of cyclic AMP-induced gene expression and differentiation. Both the receptor- and G-protein-mediated stimulation of adenylate cyclase and guanylate cyclase were unaltered in mutant fgdC as compared to wild-type cells. In wild-type cells phosphoinositi dase C was activated about twofold by the cyclic AMP receptor. In mutant fgdC cells, however, the enzyme was inhibited by about 60%. These results suggest that phosphoinositidase C is regulated by a receptor-operated activation/inhibition switch that is defective in mutant fgdC. We conclude that activation of phosphoinositidase C is essential for Dictyostelium development.
Protein myristoylation in human mononuclear phagocytes: modulation by interferon-y and tumor necrosis factor
Authors: Poli Giovanni , Sorio Claudio , Berton Giorgio
Published: April 2021

Labelling of cells with [3H]myristic acid and analysis of labelled proteins by SDS-PAGE and fluorography, enabled the identification of a limited number of myristoylated proteins in human monocytes and monocyte-derived macrophages. In human monocytes, cultivated for one to three days, major myristoylated proteins observed were of 18 kDa, 44 kDa, 60-62 kDa, 90 kDa, and a doublet of 3840 kDa. Differentiation of monocytes to macrophages by in vitro cultivation was accompanied by a selective decrease in the 60-62 kDa protein. Cultivation of the cells in the presence of the macrophageactivating cytokines interferon-y (IFN-y) and tumor necrosis factor-γ (TNF-γ), prevented the decrease in the expression of the 60—62 kDa myristoylated protein. The effect of cytokines was observed when monocytes were treated with IFN-γ or TNF-α for 24 or 48 h and protein myristoylation analyzed at day four of culture. Maintenance of monocytes in culture for up to nine days in the presence of cytokines prevented the decrease in the expression of the 60-62 kDa myristoylated protein. IFN-γ had additional effects on myristoylation of macrophage proteins. Treatment of monocytes with IFN-y for a few hours caused the induction of a 66 kDa protein. Induction of this myristoylated protein by IFN-γ was time-dependent and peaked at six hours. Analysis of the subcellular distribution of the 66 kDa protein induced by IFN-γ showed that, analogously to other myristoylated proteins, most of it was associated with cell membranes. Initial attempts to identify the myristoylated proteins in human mononuclear pha-gocytyes showed that p60src is one of the myristoylated proteins which is down-modulated during differentiation of monocytes to macrophages, and that cultivation of monocytes in the presence of IFNγ and TNF-α prevents this phenomenon.
Temporal control of differentiation by the cell nucleus: evidence for a species-specific nuclear suppression of enzymes at the early generative phase in Acetabularia
Authors: Bannwarth Horst , Groot Gon J. De
Published: April 2021

The role of the cell nucleus in controling steps of differentiation has been revealed by comparing nucleate and anucleate cells of Acetabularia (Berger et al. 1987; Bonotto, 1988). In all cases investigated so far, events related to the generative phase of the life cycle are expressed earlier in anucleate cells. Consequently, the existence of a nuclear suppressor delaying the onset of generative functions in the intact cell has been postulated (Bannwarth and Schweiger, 1983). In order to determine whether this nuclear action is species-specific, the nucleus of the species Acetabularia acetabulum was replaced by a nucleus of the species Acetabularia crenulata. The foreign nucleus did not suppress the increase in dCMP deaminase (EC 3.5.4.12) enzyme activity. This result indicates that the suppression by the nucleus is species-specific.
Normal Chlamydomonas nuclear gene structure on linkage group xix
Authors: Schloss Jeffery A. , Croom Henrietta Brown
Published: April 2021

The unusual Chlamydomonas linkage group XIX - called the uni linkage group for the uni mutants that lack one of the paired flagellae of wild-type cells - has been reported to be physically located exclusively at the basal bodies. To learn whether the structure of genes on this linkage group differs from the structure of nuclear genes in this organism, we determined the primary structure of a gene that maps to linkage group XIX. This analysis reveals the presence of nine intervening sequences; the nucleotides at exon/intron boundaries conform with nuclear gene intron junction sequences. Also typical for C. reinhardtii nuclear genes are the position and sequence of the putative polyadenylation signal. These findings suggest that transcripts from linkage group XIX are likely to be processed in the nucleus. The open reading frame, which displays weak but easily detected Chlamydomonas codon bias, potentially encodes a protein similar to a membrane anchor for cytoskeletal proteins. The observation that expression of this gene is regulated during interphase and in gametes is not consistent with the hypothesis that linkage group XIX may be expressed only during mitotic and meiotic processes.
Deoxyguanosine enhances the cytotoxicity of the topoisomerase i inhibitor camptothecin by reducing the repair of double-strand breaks induced in replicating dna
Authors: Squires Shoshana , Ryan Anderson J. , Strutt Helen L. , Smith Paul J. , Johnson Robert T.
Published: April 2021

Deoxyguanosine (dG) enhances the S phase cytotoxicity of camptothecin (CPT), a topoisomerase I (topo I) inhibitor, but by contrast does not affect the toxicity of VM26, a topoisomerase II inhibitor. The 80% survival of S phase human fibroblasts after a 60 min exposure to 0.2 pM CPT is reduced by half in the presence of 25 μM dG. Gi cells are resistant to CPT toxicity, though the levels of the single-strand DNA breaks induced by the drug are similar in Gi and S phase cells. Higher concentrations of dG retard the recovery of RNA and DNA synthesis and inhibit recovery from the S-G2 cycle block after CPT removal. At 100 μM dG the number of CPT-induced protein-linked single-strand DNA breaks is almost doubled, suggestive of a direct effect of dG on the cellular activity of topo I. In the presence or absence of dG, single-strand breaks disappear within minutes of the removal of CPT.

We found that the inhibition of topo I by CPT induces the formation of double as well as singlestrand breaks in the chromosomal DNA. Previously we have shown, using a pulse-field gel electrophoresis technique, that the double-strand breaks (DSBs) are generated predominantly at sites of replication and not in the bulk DNA. A number of these DSBs are long-lived. The present study shows that dG affects the repair of these DSBs in a dose-dependent manner, and that a higher proportion of the initial lesions induced in nascent DNA remain 24 h after removal of CPT. We suggest that the long-lived double-strand breaks, formed in replicating DNA at the time of CPT exposure, are the lethal drug-induced lesions, which explains both the selective cytotoxicity of CPT towards S phase cells and the enhancement of CPT cytotoxicity by dG.
Analysis of p53 expression in human tumours: an antibody raised against human p53 expressed in Escherichia coli
Authors: Midgley C. A. , Fisher C. J. , Bártek J. , Vojtések B. , Lane D. , Barnes D. M.
Published: April 2021

A cDNA encoding the complete normal human p53 protein was expressed in Escherichia coli using an expression system based on the bacteriophage T7 promoter. The cDNA was adapted so that the full-length protein was produced without fusion to any other sequence. Large amounts of the protein were isolated and the purified protein used to produce very high titre polyclonal antibodies to p53. These new antibodies permit the sensitive detection of p53 and p53 complexes in ELISA and immunoblotting assays. Most importantly, they also permit the detection of p53 in archival tumour material that has been conventionally fixed in formalin and embedded in paraffin wax. Using this reagent we have found that aberrant expression of p53 is a frequent feature of human breast cancer. We are able to recognise six different classes of p53 expression pattern that may be of help in the subclassification of breast tumours.
Enhanced extracellular matrix production and mineralization by osteoblasts cultured on titanium surfaces in vitro
Authors: Groessner-Schreiber Birte , Tuan Rocky S.
Published: April 2021

Long-term stability of orthopaedic and dental implants depends on the integration of the artificial material into the surrounding bone tissue. The physical and chemical properties of implants, including those made of metals such as titanium, are thought to influence osseointeg-ration. Despite the known importance of this interface, little is known about the factors that promote its formation. In this study, chick embryonic calvarial osteoblasts were cultured in vitro on smooth, rough-textured and porous-coated titanium surfaces and examined for morphology, biosynthesis of extracellular matrix and mineralization as a function of culture time. Scanning electron microscopy revealed that osteoblasts adhered securely to the titanium surface and frequently bridged the uneven surface by means of cellular processes. The osteoblast phenotype was retained in the cell cultures on titanium. In addition, the synthesis of extracellular matrix and subsequent mineralization were both substantially enhanced in the cultures on rough-textured and porous-coated titanium. These results strongly suggest that porous or rough titanium implant surfaces may act like “natural” substrata to permit microscopic tissue/cell ingrowth to improve clinical implant fixation.
Distribution of the myosin i-like ninaC proteins in the Drosophila retina and ultrastructural analysis of mutant phenotypes
Authors: Hicks Jennifer L. , Williams David S.
Published: April 2021

The Drosophila ninaC gene encodes for two head-specific proteins of 132 kDa and 174 kDa. Their predicted amino acid sequences indicate that they may have myosin I and kinase properties. We have: (1) determined the cellular and subcellular distributions of the ninaC proteins in the Drosophila retina by electron microscopic immunocytochemistry with an antibody specific for epitopes shared by both proteins; (2) characterized the ultrastructure of the mutant phenotype. The proteins were detected only in the photoreceptor cells, but were detected in all classes of the compound eye photoreceptors. Within the photoreceptors, they were found in the rhabdomeral microvilli and the cytoplasm adjacent to the rhabdo-meres. This distribution coincides with that shown previously for actin filaments. Immunolabelling of tissue from the ninaC P221 mutant, which lacks the 174 kDa protein, and two mutants whose rhabdomeres degenerate, suggests that the 132 kDa protein is present primarily in the cytoplasm adjacent to the rhabdomeres, and that the 174 kDa protein is concentrated in the rhabdomeres. Our ultrastructural analysis showed that the axial cytoskeleton of the rhabdomeral microvilli (which contains filamentous actin) was absent in both the null and P221 mutants. In the photoreceptor cell cytoplasm, the number of multivesicular bodies in the null mutant, but not the P221 mutant, was 3-fold greater in comparison with wild-type. Together, the immunolabelling results and the ultrastructural observations of the mutant phenotypes are consistent with the 174 kDa protein functioning as a component of the microvillar cytoskeleton (perhaps in an analogous manner to the 110 kDa brush border myosin I), and the 132 kDa protein playing a role in the adjacent cytoplasm by effecting some aspect of photoreceptor membrane turnover.
The role of primer recognition proteins in DNA replication: inhibition of cellular proliferation by antisense oligodeoxyribonucleotides
Authors: Kumble Krishnanand D. , Iversen Patrick L. , Vishwanatha Jamboor K.
Published: April 2021

Primer recognition proteins (PRP) are cofactors for DNA polymerase α and may have a role in laggingstrand DNA replication. PRP is composed of two subunits, which we have previously identified as the protein-tyrosine kinase substrate annexin U and phosphoglycerate kinase (PGK). In this study, we have examined the physiological involvement of these proteins in DNA synthesis and cell proliferation. When exponentially growing human HeLa cells are exposed to antisense phosphorothioate oligodeoxynucleotides to annexin H, ongoing DNA synthesis is reduced. The extent of reduction with antisense oligodeoxynucleotide to PGK was much less than with the antisense annexin n oligodeoxy nucleotide. Reductions in the labeling and mitotic indices of HeLa cell cultures are seen after exposure to antisense oligodeoxynucleotides. Flow cytometric analyses indicate that progression from S phase to G2 phase of the cycle is retarded by exposure of cells to the antisense oligodeoxynucleotides. Corresponding sense oligodeoxynucleotides have no inhibitory effects on these parameters. The new synthesis of annexin H and PGK is specifically reduced in the presence of antisense oligodeoxynucleotides, indicating that the complex of newly synthesized annexin H and PGK may participate in PRP function. These experiments indicate that annexin H and PGK may have a physiological role in DNA synthesis and cell cycle progression, and represent the first physiological role for annexin H monomer in cells.
The increase in intracellular ph associated with Xenopus egg activation is a ca2+-dependent wave
Authors: Grandin Nathalie , Charbonneau Michel
Published: April 2021

In Xenopus eggs, the transient increase in intracellular free calcium ([Ca2+]i), or Ca2+ transient, which occurs 1–3 min after egg activation, is likely to be partly responsible for the release of the cell cycle blockade. In the present study, we have used microinjection of BAPTA or EGTA, two potent chelators of Ca2+, to buffer [Ca2+] i at various steps during Xenopus egg activation and evaluate the impact on some of the associated events. Microinjection of either one of the Ca2+ chelators into unactivated eggs prevented egg activation without, however, lowering [Ca2+]i, suggesting that only physiological [Ca2+]i changes, but not [Ca2+]1 levels, were affected by the Ca2+ buffer. When BAPTA was microinjected around the time of occurrence of the Ca2+ transient, the egg activation-associated increase in intracellular pH (pH0 was clearly delayed. That delay was not due to a general slowing down of the cell cycle, since under the same conditions of microinjection of BAPTA the kinetics of MPF (a universal M-phase promoting factor) inactivation were unaffected. These results represent the first indication that the Ca2+ transient participates in determining the time of initiation of the pH1 increase during Xenopus egg activation. The present results also demonstrate that the egg activation-associated pHi changes (a slight, transient decrease in pH, followed by a permanent increase in pH|) proceed as a wave propagating from the site of triggering of egg activation. Experiments of local microinjection of BAPTA support the view that the pH wave is a consequence of the Ca2+ wave, which it follows closely.
Free ca2+ gradient in growing pollen tubes oflilium
Authors: Miller Deborah D. , Callaham Dale A. , Gross David I. , Hepler Peter K.
Published: April 2021

Fluorescence ratiometric imaging of Lilium pollen tubes loaded with the Ca2+ indicator Fura-2 dextran has revealed a distinct elevation of free intracellular calcium ion concentration ([Ca2+]i) at the extreme tip of actively growing Lilium pollen tubes that declines to a uniform basal level of ∼170 nM throughout the length of the tube. The calcium gradient occurs within the first 10–20 μM proximal to the tip. Experimental inhibition of tip growth, usually achieved through the injection of the Ca2+ buffer 5,5’-dibromo BAPTA, results in the loss of the [Ca2+]igradient. Occasionally these inhibited cells reinitiate growth, and when they do so ratio imaging reveals that the tip gradient of free [Ca2+Ji re-emerges. The results presented here are very different from those previously published by revealing the presence of the [Ca2+]i gradient that is restricted to the 10–20 μM adjacent to the tube tip. Further, these experiments demonstrate a strict correlation between the presence of a [Ca2+Ji gradient, and tip growth in Lilium pollen tubes.
Disruption of synchrony between parasite growth and host cell division is a determinant of differentiation to the merozoite in Theileria annulata
Authors: Shiels Brian , Kinnaird Jane , Mckellar Susan , Dickson Joanne , Miled Leila Ben , Melrose Ron , Brown Duncan , Tait Andy
Published: April 2021

The multinucleated macroschizont stage of the proto-zoon Theileria annulata is an intracellular parasite of bovine leukocytes. The parasite induces the host cell to proliferate, and divides in synchrony with the immortalised host cell. Differentiation to the next stage occurs within the host cell culminating in the release of merozoites and destruction of the leukocyte. In this study clones of Theileria annulata macroschizont-infected cell lines were isolated by limiting dilution and tested for differentiation to the merozoite stage (mero-gony). Two cloned cell lines underwent differentiation with enhanced efficiency, while two others were of lower efficiency. Quantification was carried out using monoclonal antibodies, which showed that over 90% of the cells in an enhanced cloned cell line could be induced to differentiate. By carrying out induction at 41°C for limited periods of time followed by culture at 37°C evidence was obtained that differentiation to the merozoite is a two-step process: a preliminary reversible phase, followed by a second irreversible phase of differentiation. Analysis of the nuclear number of the macroschizont and the growth rate of the cloned cell lines showed that the ability to differentiate was associated with an increase in nuclear number (size) of the macroschizont, generated by a disruption in the synchrony between parasite growth and host cell division. We believe that these results reveal a relationship between a reduction in parasite division and differentiation, and that there are similarities between stage differentiation in parasites and cellular differentiation in higher eukaryotes.
Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles : Implications for the role of microtubule dynamics in mitosis
Authors: Ann Jordan Mary , Thrower Douglas , Wilson Leslie
Published: April 2021

Inhibition of mitosis by many drugs that bind to tubulin has been attributed to depolymerization of microtubules. However, we found previously that low concentrations of vinblastine and vincristine blocked mitosis in HeLa cells with little or no depolymerization of spindle microtubules, and spindles appeared morphologically normal or nearly normal. In the present study, we characterized the effects of vinblastine, podophyllotoxin and nocodazole over broad concentration ranges on mitotic spindle organization in HeLa cells. These three drugs are known to affect the dynamics of microtubule polymerization in vitro and to depolymerize microtubules in cells. We wanted to probe further whether mitotic inhibition by these drugs is brought about by a more subtle effect on the microtubules than net microtubule depolymerization. We compared the effects of vinblastine, podophyllotoxin and nocodazole on the organization of spindle microtubules, chromosomes and centrosomes, and on the total mass of microtubules. Spindle organization was examined by immunofluorescence microscopy, and microtubule polymer mass was assayed on isolated cytoskeletons by a quantitative enzyme-linked immunoadsorbence assay for tubulin. As the drug concentration was increased, the organization of mitotic spindles changed in the same way with all three drugs. The changes were associated with mitotic arrest, but were not necessarily accompanied by net microtubule depolymerization. With podophyllotoxin, mitotic arrest was accompanied by microtubule depolymerization. In contrast, with vinblastine and nocodazole, mitotic arrest occurred in the presence of a full complement of spindle microtubules. All three drugs induced a nearly identical rearrangement of spindle microtubules, an increasingly aberrant organization of metaphase chromosomes, and fragmentation of centrosomes. The data suggest that these anti-mitotic drugs block mitosis primarily by inhibiting the dynamics of spindle microtubules rather than by simply depolymerizing the microtubules.
Cell cycle events in the green alga Chlamydomonas eugametos and their control by environmental factors
Authors: Zachleder V. , Ende H. Van Den
Published: April 2021

A procedure for routine synchronization of large amounts of the unicellular green alga Chlamydomonas eugametos in liquid culture by alternating light and dark periods is described. The synchronized populations were grown at various light intensities and temperatures. The effect of these variables on the lengths of parts of the cell cycle and the number of daughter cells per cell division was followed. The cell cycle of C. eugametos started with a period in which the cells increased in size only (precommitment period). The length of this period was dependent on both the light intensity and the temperature. At the end of this period, a key point of the cell cycle (called commitment point) was attained. From this point, the cell were committed to divide and cell reproduction was triggered. The following period (postcommitment period), during which daughter cells were formed, could be traversed without supply of external energy, and without further growth of the cells. However, if sufficient energy was supplied during this period, the cells were able to attain more commitment points, leading to a higher number of daughter cells. The postcommitment period was fairly constant within a certain range of light intensity. At light intensities leading to more commitment points, however, this period was prolonged. No evidence was found for circadian rhythms or endogenous factors of “Zeitgeber” type playing a role in the control of growth and reproductive sequences in the cell cycle of C. eugametos.
Autophagic vacuoles rapidly fuse with pre-existing lysosomes in cultured hepatocytes
Authors: Lawrence B. Paige , Brown William J.
Published: April 2021

Autophagic vacuoles (A Vs) arise when membranes of the ER sequester parts of the cytoplasm, forming a new, double-membraned vacuole, to which lysosomal enzymes are then delivered. To investigate the mechanism of lysosomal enzyme delivery to nascent AVs, amino acid (AA) starvation and glucagon treatment were used to induce autophagy in a cultured cell system using rat hepatocytes (Fu5C8 cells). The induction of autophagy was assayed using biochemical, morphometric and immunocytochemical techniques. In these cells, AA starvation resulted in a fivefold increase in total cellular proteolysis, and sixfold and 4.5-fold increases in the volume and surface densities of AVs, respectively. Using an antibody against the mannose 6-phosphate receptor (MPR) and two sizes of colloidal gold to label separately and track the endosomal and lysosomal compartments, the time course of endosomal and lysosomal fusion with AVs was analyzed in detail. On the basis of these experiments, we found that AVs rapidly fuse with preexisting lysosomes, but seldom with a prelysosomal compartment (PLC). Using immunoperoxidase, staining for the MPR was infrequently observed in association with any AVs. However, at early times following the induction of autophagy (<2 h), many autophagic vacuoles stained positively for the lysosomal enzyme cathepsin D. Consistent with these results, treatment of cells with tunicamycin had no effect on autophagy-induced proteolysis. We conclude that lysosomal enzyme delivery to nascent AVs occurs primarily by the fusion of pre-existing mature lysosomes, with a much smaller contribution by MPRs or the PLC.
Characterization of the enterocyte-like brush border cytoskeleton of the C2BBe clones of the human intestinal cell line, Caco-2
Authors: Peterson Michelle D. , Mooseker Mark S.
Published: April 2021

The brush border (BB) of the enterocyte is a well-studied example of the actin-based cytoskeleton. We describe here a cell culture model that expresses a faithful representation of the in vivo structure. Two clones (C2BBe 1 and 2) isolated from the cell line Caco-2 (derived from a human colonic adenocarcinoma) formed a polarized monolayer with an apical BB morphologically comparable to that of the human colon. BBs could be isolated by standard methods and contained the microvillar proteins villin, fimbrin, sucrase-isomaltase and BB myosin I, and the terminal web proteins fodrin and myosin II. The immunolocalization of these proteins in confluent, filter-grown monolayers was determined by laser scanning confocal microscopy; patterns of distribution comparable to those in human enterocytes were observed. Sedimentation analysis of cell homogenates derived from C2BBe cells and human colonic epithelial cells demonstrated similar patterns of fractionation of BB proteins; the physical association of those proteins, as determined by extraction from the BB, was also comparable between the two cell types. Like enterocytes of the human intestine, C2BBe cells expressed multiple myosin I immunogens reactive with a head domainspecific monoclonal antibody raised against avian BB myosin I, one of which co-migrated with the ∽ 110 kilodalton (kDa) heavy chain of human BB myosin I. In addition, the C2BBe cells express a pair of higher molecular mass immunogens (130 and 140 kDa). These myosin I immunogens all exhibit ATP-dependent association with the C2BBe cytoskeleton. Although the higher molecular mass immunogens were detected in several other human intestinal lines examined, including the parent Caco-2 line, none of these other lines expressed detectable levels of the 110 kDa immunogen, which is presumed to be the heavy chain of human BB myosin I.
Actin has multiple roles in the formation and architecture of zoospores of the oomycetes, Saprolegnia ferax and Achlya bisexualis
Authors: Heath I. Brent , Harold Ruth L.
Published: April 2021

Very similar changing patterns of actin are described with rhodamine-phalloidin labelling during the zo-osporic life cycle of the oomycetes, Saprolegnia ferax and Achlya bisexualis. By comparing the changes with previously described ultrastructural and functional changes, we show that actin functions in numerous previously unrecognized processes.

Most spectacularly, the directed vesicle expansions of the cytokinetic system involve newly formed actin which outlines the developing zoospores. Disruption of this actin with cytochalasins leads to abnormal cleavage as witnessed by the formation of enlarged and irregular cysts. Prior to cytokinesis, two new types of organelle are synthesized and one, known as K bodies, clusters around the nuclei. These organelles are actin-rich during development and clustering, consistent with actin functioning in their positioning.

In the zoospores, actin is concentrated around the water expulsion vacuoles, indicating that they are contractile, and permeates the cytoplasm, probably with a skeletal role. This concept is supported by the first demonstration of actin specifically associated with a microtubular root in the secondary zoospore. Upon encystment there is a dramatic increase in stained actin in the form of peripheral plaques associated with the newly synthesized cell wall.

When the cysts germinate, a fibrillar actin cap, comparable to that previously described in hyphal tips, forms in the germ tube apex, but only after cell wall softening to permit germ tube protrusion. This sequence is consistent with the actin cap modulating turgor-driven expansion of the tip as previously discussed for hyphae.

In addition to disrupting cleavage-associated actin, cytochalasins show developmental stage, dose and drug (CE>CD>CB) specific effects on zoosporulation-related actin, which indicates that, contrary to previous suggestions, rhodamine-phalloidin staining is a useful indicator of actin behaviour in response to cytochalasins. These responses include differential effects on adjoining actin arrays, some of which are transient in the continued presence of the drugs, indicating a mechanism of drug adaptation.
The localization of myosin I and myosin II in Acanthamoeba by fluorescence microscopy
Authors: Yonemura Shigenobu , Pollard Thomas D.
Published: April 2021

We used several fixation protocols and a panel of monoclonal antibodies to re-examine the localization of myosin I and myosin II in Acanthamoeba. Two monoclonal antibodies that bind to the head of myosin II stain a range of particles in the cytoplasm. The smallest and most numerous cytoplasmic particles are about the same size and intensity as myosin II minifilaments and are distributed throughout the endoplasm. The largest particles stain like myosin II thick filaments and are concentrated in the cleavage furrow of dividing cells and in the tail of locomoting cells. Five different monoclonal antibodies that bind to the myosin II tail also stain cytoplasmic particles but with a limited range of intensity. None of the myosin II monoclonal antibodies stains the contractile vacuole or plasma membrane. Two monoclonal antibodies to myosin I gave punctate cytoplasmic staining that did not correspond clearly to any of the phase-dense particles in the cytoplasm. In many, but not all, locomoting cells, the myosin I staining was concentrated at the leading edge. Both myosin I antibodies stained a single cytoplasmic vacuole of variable size that was presumed to be the contractile vacuole. The antibody that binds myosin IA but not myosin IB stained novel intercellular contacts and the antibody that binds both myosin IA and myosin IB stained the plasma membrane, especially the tips of filopodia.
Changes in cell surface expression of fibronectin and fibronectin receptor during liver regeneration
Authors: Pujades Cristina , Forsberg Erik , Enrich Carlos , Johansson Staff An
Published: April 2021

The surface expression of fibronectin and its major integrin receptor in liver, integrinα5β1 was studied during liver regeneration after partial hepatectomy. Using immunoblotting, plasma membranes isolated from livers at different regeneration stages were found to contain 6- to 8-fold elevated levels of fibronectin, α5 and β1 at 12-24 h after the operation. Normal levels were gradually restored during the following 8-9 days. The membrane-associated fibronectin lacked the ED-A domain, suggesting that it consisted of plasma fibronectin. A prominent fibronectin fragment (180 kDa) was present at 12-24 h after surgery, possibly reflecting turnover of the pericellular matrix during cell division.

Indirect immunohistochemical staining of liver sections revealed β1 and fibronectin mainly in the sinusoidal region of the hepatocyte plasma membrane. The distribution was not markedly altered during liver regeneration. The results suggest that the fibronectin-mediated contacts between the cells and the extracellular matrix increase during the pre-replicative and proliferative phases of liver regeneration. The significance of these results for the growth and for the structure of the liver during regeneration and development is discussed.