bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of mitosis by okadaic acid: possible involvement of a protein phosphatase 2A in the transition from metaphase to anaphase
Authors: Vandré Dale D. , Wills Victoria L. 
Published: April 2021

ABSTRACT
The effects of the protein phosphatase inhibitor okadaic acid were examined using the pig kidney cell line LLC-PK. At relatively low concentrations of the inhibitor (8–40 μM), cells became blocked in a metaphase-like mitotic state beginning 6–8 h after initial treatment. Spindle microtubules were present throughout the period of the mitotic block, but were not stabilized since they remained sensitive to nocodazole depolymerization. With increasing length of the mitotic block chromosome alignment at the metaphase plate was disrupted and multipolar spindles developed. Cells continued to accumulate in mitosis for at least 24 h, indicating that at these low concentrations okadaic acid was not cytotoxic, but rather acted as a cytostatic agent. Upon release of the okadaic acid block, mitotic LLC-PK cells recovered and completed anaphase. After extended periods of treatment some cells were able to escape the okadaic acid-induced mitotic block. These cells were multinucleate and had undergone cytokinesis in the absence of chromosome segregation. At higher concentrations of okadaic acid (0.5–1.0 μM), mitosis was blocked within 30-60 min of treatment. However, within 90–120 min treated cells rounded up and detached from the monolayer, regardless of whether they were in interphase or mitosis. Cytoplasmic microtubules were depolymerized in the detached cells, and these cells could not recover from the cytotoxic effects of such high concentrations of okadaic acid. Thus, differential effects of the phosphatase inhibitor could be demonstrated, depending upon the concentration of okadaic acid applied to the cultures. The okadaic acid-induced mitotic blockage was probably due to the inhibition of a type 2A protein phosphatase that is involved in the transition from metaphase to anaphase.
Cell cycle-specific disruption of the preprophase band of microtubules in fern protonemata: effects of displacement of the endoplasm by centrifugation
Authors: Murata Takashi , Wada Masamitsu 
Published: April 2021

ABSTRACT
The preprophase band (PPB) of microtubules (MTs), which appears at the future site of cytokinesis prior to cell division in higher plant cells, disappears by metaphase. Recent studies have shown that displacement of the endoplasm from the PPB region by centrifugation delays the disappearance of the PPB. To study the role of the endoplasm in the cell cycle-specific disruption of the PPB, the filamentous protonemal cells of the fern Adiantum capiUus-veneris L. were centrifuged twice so that the first centrifugation displaced the endoplasm from the site of the PPB and the second returned it to its original location. The endoplasm, including the nucleus of various stages of mitosis, could be returned by the second centrifugation to the original region of the PPB, which persists during mitosis in the centrifuged cells. When endoplasm with a prophase nucleus was returned to its original location, the PPB was not disrupted. When endoplasm with a prometaphase-telophase nucleus was similarly returned, the PPB was disrupted within 10 min of termination of centrifugation. In protonemal cells of Adiantum, a second PPB is often formed near the displaced nucleus after the first centrifugation. In cells in which the endoplasm was considered to have been returned to its original location at the prophase/prometaphase transition, the second PPB did not disappear even though the initial PPB was disrupted by the endoplasm. These results suggest that cell cycle-specific disruption of the PPB is regulated by some factor(s) in the endoplasm, which appears at prometaphase, i.e. the stage at which the PPB is disrupted in non-centrifuged cells.
Subcellular imaging of calcium exchange in cultured cells with ion microscopy
Authors: Chandra Subhash , Ausserer Walter , Morrison George H. 
Published: April 2021

ABSTRACT
Calcium to calcium exchange between intracellular and extracellular pools has been imaged directly in individual cells using stable 44Ca (98.78% enrichment) in the nutrient medium and an isotopic imaging technique, ion microscopy. Observations were made by imaging mass 40 to determine the native intracellular calcium (40Ca), and mass 44 to localize the 44Ca from the extracellular medium that exchanged with cellular calcium. LLC-PK, porcine kidney epithelial cells were exposed to the nutrient medium with 1.87 mM stable 44Ca for 0,1, 2, 5, 8, 20, 60 and 90 min, and cryogenically prepared prior to ion microscopic analysis. The cell nucleus, the Golgi region and the remaining cell cytoplasm could be spatially resolved to within about 0.5 fan using the ion microscope. On the basis of the kinetics of 40Ca to 44Ca exchange it was observed that all three compartments had a rapidly exchanging pool of calcium, which took about 2 min to exchange. A moderately rapidly exchanging pool of calcium was identified between 2 and 20 min of calcium exchange. The cells had exchanged about 50% of their total internal calcium with the external calcium in less than 20 min. The remaining 50% of the cellular calcium could be classified as a slowly exchanging pool. Isotopic images of 39K and 23Na were recorded along with 40Ca and 44Ca images to assess the health status of the cell. Isotopic imaging has the unique ability to distinguish intracellular calcium from the extracellular calcium that enters cells and has enormous potential for studies of calcium transport under physiological and pathological conditions.
Differential expression of the VLA family of integrins along the crypt-villus axis in the human small intestine
Authors: Beaulieu Jean-François 
Published: April 2021

ABSTRACT
Regulation of epithelial cell proliferation, migration and differentiation under physiological conditions remains poorly understood. Interaction of the cells with their underlying basement membrane through integrins, a specific subset of cell surface binding proteins, is one potential mechanism. In the present work, I examined this hypothesis by investigating the distribution of a variety of epithelial basement membrane proteins and the expression of the members of the VLA family of integrins in the adult intestinal epithelium. Indeed, this rapidly renewing simple epithelium contains within its functional unit, the crypt-villus axis, essentially two distinct cell populations: the proliferative and undifferentiated crypt cells and the mature enterocytes on the villus. Although immunolocalization of basement membrane molecules revealed that laminin, type IV collagen and heparan sulfate proteoglycan are distributed homogeneously all along the crypt-villus axis, other nonexclusive basement membrane components were found differentially expressed. Tenascin was concentrated at the base of both villus and lower crypt cells while cellular fibronectin was mostly detected in association with the crypt cells. Moreover, VLA β;1 as well as 5 of the 6 VLA α subunits tested were expressed by intestinal epithelial cells under specific patterns of staining. The β1 and α6subunits were strongly detected at the base of all enterocytes while β5 also detected all along the cryptvillus axis, was weaker and consistently appeared with a punctated/interrupted pattern. On the other hand, the VLA α1 α2 and α3 were expressed at the basolateral domains of enterocytes under distinctive crypt-villus gradients. The α1 subunit was detected at the base of all epithelial cells but lateral staining was only observed in differentiating cells (middle and upper crypt). Finally, in most specimens, α2 and α3 displayed strictly complementary staining patterns for the lower crypt region (α2β3—) and the upper crypt-to-villus region (β2 —, β3). Taken together, these data emphasize that proliferation, migration and differentiation in the normal state are susceptible to various influences including compositional changes in the basement membrane and differential expression of receptors for these components.
Collagen-induced rapid morphogenesis of human mammary epithelial cells: The role of the α
2
β
1 integrin
Authors: Berdichevsky Feodor , Gilbert Chris , Shearer Moira , Taylor-Papadimitriou Joyce 
Published: April 2021

ABSTRACT
The cell line MTSVl-7, originally derived by immortalizing mammary epithelial cells cultured from human milk was able to form three-dimensional structures in collagen gel. We have now found that these cells, cultured as a monolayer, are able to undergo rapid morphogenesis forming ridges and balls around collagen fibres, when soluble collagen type I is added to the medium. Monoclonal antibodies to the α2(PlE6) and β1(mAB13) subunits of VLA-2, but not to the α3-subunit (P1B5) of VLA-3, could block this collagen-induced rapid morphogenesis (CIRM). The effect of the antibodies on cell attachment, spreading, and migration on collagen gels was analyzed to identify α2β1 dependent steps which might be involved in CIRM. The results suggest that while other proteins, besides α2α1, are also involved in cell attachment and migration, cell spreading was specifically blocked by antibodies to the VLA-2, but not to the VLA-3 integrin. The results demonstrate that the α2α1 integrin plays a crucial role in the collagen-induced morphogenesis of human mammary epithelial cells and implicate the process of VLA-2-dependent cell spreading as an important step in this morphogenesis.
Nucleolus behaviour during the cell cycle of a primitive dinoflagellate eukaryote,Prorocentrum micansEhr., seen by light microscopy and electron microscopy
Authors: Soyer-Gobillard Marie-Odile , Geraud Marie-Line 
Published: April 2021

ABSTRACTLight-microscopy observation of the dinoflagellate Prorocentrum micans after silver-staining of the argyrophilic proteins of the nucleolar organizing region (Ag-NOR staining) showed the presence of nucleolar material throughout the vegetative cell cycle, and in particular during all the mitotic stages. This contrasts with the case in most higher eukaryotes, in which nucleoli disappear at the end of prophase and are reconstituted in daughter cells during telophase.Electron-microscope (EM) observations after conventional or fast-freeze fixation revealed that during interphase several functional nucleoli with three compartments (NORs, the fibrillogranular and the preribosomal granular compartments) are present in a nucleus in which the envelope is persistent and the chromosomes are always compact. During early prophase, when chromosomes are beginning to split, the nucleoli remain functional, whereas in late prophase they contain only a NOR and the granular component, and the chromosomes are surrounded by many protein masses. In early telophase, the nucleolar material coating the chromosomes migrates along with the chromosomes. Nucleologenesis occurs through the formation of prenucleolar bodies around lateral or telomeric nucleofilaments extruding from the chromosomes. Several chromosomes can contribute to the formation of one nucleolus. The behaviour of these ‘persistent nucleoli’ in a closed-nucleus model such as that of the dinoflagellates is discussed with regard to the higher eukaryotes.
Expression and function of ras proto-oncogene proteins in human sperm cells
Authors: Naz Rajesh .K , Ahmad Khaliq , Kaplan Paul 
Published: April 2021

ABSTRACT
The presence and role of c-ras proteins were investigated in mature human sperm cells. The v-H-ras monoclonal antibody (mAh) against the c-ras protein, p21, reacted specifically with the acrosomal region of methanol-fixed as well as unfixed-live capacitated and non-capacitated human sperm cell in the indirect immunofluorescence technique. The v-H-ras mAb predominantly recognized c-ras protein of 21 kDa on the Western blot of lithium diiodosalicylate (LlS)-solubilized human sperm preparation. The incubation of sperm cells with v-H-ras mAb affected the sperm cell function in the human sperm penetration assay. The antibody significantly reduced the acrosome reaction and release of acrosin activity from the sperm cells. There was no effect of the mAb on percentage motility, although the mAb significantly affected various motility characteristics such as linearity, amplitude of lateral head displacement (ALH) and beat frequency, the motility parameters involved in the hyperactivation phenomenon of sperm cells leading to capacitation and acrosome reaction. These results suggest that the c-ras or c-ras-like proteins are present in mature sperm cell and may have a role in capacitation and/or acrosome reaction of human sperm cell.
Effect of caffeine on intracellular transport of Semliki Forest virus membrane glycoproteins
Authors: Kuismanen Esa , Jäntti Jussi , Mäkiranta Virpi , Sariola Merja 
Published: April 2021

ABSTRACT
The effect of caffeine on the intracellular transport of Semliki Forest virus (SFV) membrane glycoproteins was studied in baby hamster kidney (BHK) cells. The movement of the proteins was affected at two steps in the exocytic pathway. The exit of the proteins from the endoplasmic reticulum (ER) was inhibited by 10 mM caffeine at 20°C, a temperature that normally allows transport to the Golgi complex. At higher temperatures (28°C and 37°C) in the presence of 10 mM caffeine exit from the ER occurred, but the proteins accumulated at intracellular membrane elements. Immunofluorescence localization, endoglycosidase-H analysis, and analysis of the proteolytical cleavage of the p62 precursor protein suggested that transport in the presence of 10 mM caffeine was arrested at the membranes between the trans-Golgi and the plasma membrane.
In situ localisation of single-stranded DNA breaks in nuclei of a subpopulation of cells within regenerating skeletal muscle of the dystrophic mdx mouse
Authors: Coulton G. R. , Rogers B. , Strutt P. , Skynner M. J. , Watt D. J. 
Published: April 2021

ABSTRACT
Degeneration of muscle fibres during the early stages of Duchenne Muscular Dystrophy (DMD) is accompanied by muscle fibre regeneration where cell division and myoblast fusion to form multinucleate myotubes within the lesions appear to recapitulate the events of normal muscle development. The mechanisms that govern the expression of genes regulating differentiation of myoblasts in regenerating skeletal muscle are of great interest for the development of future therapies designed to stimulate muscle regeneration. We show here that single-stranded breaks in DNA are localised in nuclei, using an exogenously applied medium containing labelled deoxynucleotides and the Klenow fragment of DNA polymerase I. The nuclei of a sub-population of cells lying in the inflammatory infiltrate of lesions in the skeletal muscle of the muscular dystrophic mouse (mdx), a genetic homologue of DMD, were labelled in this fashion. By contrast, labelled cells were completely absent from the muscles of normal non-myopathic animals (C57BL/10) and non-lesioned areas of mdx muscles. Cells expressing the muscle-specific regulatory gene, myogenin, were also found within mononucleate cells and myotubes within similar mdx muscle lesions.
While we cannot yet say that the cells labelled by the DNA polymerase reaction are in fact differentiating, they were found only in significant numbers within mdx muscle lesions where new muscle fibres appear, providing strong circumstantial evidence that they are intimately associated with the regenerative process. Using a range of nucleases and different DNA polymerases, we show that the DNA polymerase-labelling reaction observed was DNA-dependant and most probably due to infilling of naturally occurring single-stranded gaps in DNA.
Since the regenerative process in human Duchenne Muscular Dystrophy is apparently less effective than that seen in mdx mice, continued study of singlestranded DNA breaks may help to elucidate further the mechanisms controlling the expression of genes that characterise the myogenic process during skeletal muscle regeneration. Such findings might be applied in the development of future therapies designed to stimulate muscle regeneration in human dystrophies.
Isoprenylation of rab proteins on structurally distinct cysteine motifs
Authors: Peter Matthias , Chavrier Philippe , Nigg Erich A. , Zerial Marino 
Published: April 2021

ABSTRACT
rab proteins are low molecular weight GTP-binding proteins highly related to Yptlp and Sec4p, which are involved in the control of secretion in yeast Saccharo-myces cerevisiae. Morphological and biochemical studies have shown that rab proteins are membrane associated and are localized to specific subcompartments along the exocytic and endocytic pathway. Membrane association requires the presence of C-terminal cysteine residues. The present report indicates that the structurally distinct cysteine motifs of rab proteins are subjected to isoprenylation both in vitro and in vivo. Studies on deletion mutants suggest that an intact C-terminal end is required for the association of rab proteins with the membrane and is necessary for the post-translational modification. Finally, we show that the isoprenoid transferase which modifies rab termini is different from the enzyme which farnesylates nuclear lamins and ras proteins in vitro.
Immunolocalization of kinesin in sea urchin coelomocytes: Association of kinesin with intracellular organelles
Authors: Henson John H. , Nesbitt David , Wright Brent D. , Scholey Jonathan M. 
Published: April 2021

ABSTRACT
We have recently used domain-specific monoclonal antibodies (mAbs) to immunofluorescently localize kinesin to vesicle-like structures in the cytoplasm of sea urchin coelomocytes. In order to characterize further these localization patterns we have examined the distribution of kinesin with respect to the arrangement of microtubules (MTs) and various organelles. In double-label experiments involving the immunofluorescent staining of kinesin (using a mixture of the mAbs SUK2, 4 and 5), MTs were labeled with an antiserum against sea urchin tubulin, the endoplasmic reticulum (ER) was labeled with an antiserum against a luminal calsequestrin-like protein, the Golgi apparatus was labeled with rhodamine-wheat germ agglutinin (WGA) or NBD-ceramide, mitochondria were labeled with rhodamine 123, endosomes were labeled with Texas Red-ovalbumin, and lysosomes were labeled with Lucifer yellow or acridine orange. Kinesin-labeled vesicle-like structures were found in the same regions of the cells as MTs and the ER, being widely distributed in motile cells, but restricted to the perinuclear regions of stationary cells. There also appeared to be a correlation between the distribution of endosomes and kinesin staining in a sub-population of cells. The kinesin binding structures were found occasionally to align in linear arrays, consistent with the idea that kinesin may transport ER and endosomes along linear MT tracks. No clear correlations were observed between the kinesin staining and the distribution of mitochondria, the Golgi apparatus or lysosomes, suggesting that kinesin may specifically associate with only a subclass of organelles in coelomocytes.
Involvement of intracellular calcium in protein secretion in Dictyostelium discoideum
Authors: Coukell M. B. , Cameron A. M. , Adames N. R. 
Published: April 2021

ABSTRACT
We reported previously that Ca2+ depletion of Dic tyostelium discoideum cells severely inhibits extracellular cyclic nucleotide phosphodiesterase (PD) synthesis at a post-transcriptional step. In this study, further experiments were performed to learn more about the nature of this phenomenon. Examination of the polysomal distribution of PD transcripts in control cells and in cells depleted of Ca 2+ by incubation with EGTA and A23187 (EA) suggested that inhibition of PD production does not involve translational control. Kinetic analysis of this inhibitory process revealed that soluble, intracellular PD activity, synthesized from either the 2.4 or 1.9 kb PD mRNA, decreased very rapidly upon addition of EA. Furthermore, this decrease in activity was accompanied by the preferential loss of PD-related polypeptides, indicating a proteolytic event. EA-induced PD degradation required cellular energy and concomitant protein synthesis but was unaffected by most of the lysosomotropic agents tested. Therefore, PD proteolysis might not occur in the lysosome. In cell fractionation experiments, the EA-sensitive, intracellular PD activity comigrated with a rough ER marker in Percoll/KCl gradients. In addition to its effect on the PD, EA were also observed to inhibit production and rapidly lower the intracellular levels of another secreted glycoprotein, the PD inhibitor. Together, these results suggest that depletion of some intracellular Ca2+ store(s) in Dictyostelium, possibly the ER, disrupts the normal function of the secretory pathway, resulting in selective degradation of certain proteins.
Inhibition by kinase inhibitors of the development and the disappearance of the preprophase band of microtubules in tobacco BY-2 cells
Authors: Katsuta Junko , Shibaoka Hiroh 
Published: April 2021

ABSTRACT
The effects of two kinase inhibitors, staurosporine and K-252a, on the development and the disappearance of the preprophase band of microtubules (PPB) were studied in tobacco BY-2 cells, the cell cycle of which had been synchronized. Treatment of cells at late G2 phase or at prophase with 20 M staurosporine or 2 M K-252a for 2 or 3 h caused a decrease in the rate of development of PPBs and inhibited the disappearance of PPBs once they had developed, with the resultant accumulation of cells with a PPB.
Cortical microtubules (MTs) or PPBs on protoplast ghosts, which were prepared from BY-2 cells in late G2 phase or at prophase, disappeared on treatment of the ghosts with ATP or ATP S but not on treatment with AMP-PNP. The effect of ATP on the disappearance of MTs on the ghosts was suppressed by 20 M staurosporine. Although 2 M K-252a applied to the culture medium suppressed the anticipated disappearance of PPBs, it did not suppress the disappearance of MTs on the ghosts that was caused by ATP. We propose tentatively that a kinase sensitive to staurosporine but not to K-252a is directly involved in the disappearance of MTs and that a kinase that is sensitive to K-252a is indirectly involved in this process.
The disappearance of MTs on protoplast ghost that was induced by ATP did not occur in the presence of 20 μM taxol.
Variant choriocarcinoma (BeWo) cells that differ in adhesion and migration on fibronectin display conserved patterns of integrin expression
Authors: Aplin John D. , Sattar Abdul , Mould A. Paul 
Published: April 2021

ABSTRACT
Starting from the BeWo choriocarcinoma cell line, two stable variant cell lines (epi and lc) were isolated. Epi cells displayed an epithelioid colony morphology while lc were fibroblastoid. lc cells attached and spread on fibronectin-coated surfaces at significantly lower density of fibronectin than epi or the parent cell line. lc also migrated more efficiently to fibronectin in a trans-filter assay than either epi or parent cells. Integrin expression by the cell lines was investigated by flow cytometry and immunoprecipitation from surface-labelled cells with a panel of subunit-specific antibodies. Integrins α2β1, α5β1, αvβ1 and α6β4 were detected in each case, and levels of expression were identical in the two variant lines. Anti-functional antibodies were used to probe the role of integrins in fibronectin- and vitronectin-medi-ated adhesion. Complete inhibition of adhesion to fibronectin was observed with anti-β1 antibody, and par-tial inhibition with anti-α5, suggesting that integrin α5β1 is mainly responsible for the interaction. Adhesion to vitronectin was inhibitable using anti-αv and anti-β1 antibodies, suggesting that integrin αvβ1 is active in these cells as a vitronectin receptor. There was a correlation between the altered morphology of the variant cells and alterations in the distribution of integrin α6β4 and laminin in monolayer cultures. The results support the idea that fibronectin may mediate the migratory behaviour of extravillous trophoblast in vivo. Switch to a more migratory phenotype may be mediated by the selective activation of integrins and altered interaction with basement membrane.