bluuebunny/crossref_metadata_2025_split huggingface dataset:
Three-dimensional analysis of the organization of human chromosome domains in human and human-hamster hybrid interphase nuclei
Authors: Dekken Herman Van , Pinkel Daniel , Mullikin Jim , Trask Barbara , Engh Ger Van Den , Gray Joe 
Published: April 2021

ABSTRACT
This report describes the intranuclear organization of chromosomes in human–hamster hybrid nuclei and in human cell nuclei. The target chromosomes were stained using in situ hybridization with biotin-ylated, chromosome-specific DNA probes. Bound probe was detected with fluorescein–avidin. Hybridizations were performed to fixed nuclei in aqueous suspension in order to preserve their threedimensional morphology. Total nuclear DNA was stained with DAPI. Three-dimensional information about the organization of DNA and probe within the nucleus was obtained by optical sectioning.
The human chromosomes in human–hamster hybrid nuclei were found to be confined to ‘domains’ that were maintained during the cell cycle. Different spatial localization patterns of the human chromosomes were seen in interphase nuclei of two different hybrid cell lines. The positions of chromosome-specific repetitive sequences in human fibroblast interphase nuclei were also studied using probes for the telomeric region of chromo-some Ip (Ip36), the centromeric region of chromosome 9 (9ql2) and the long arm of the Y chromosome (Yql2). These studies showed that the two lρ telomeric loci are located near the nuclear surface. The chromosome 9 centromeric loci are similarly located. Simultaneous hybridization of the chromosome 1 telomeric probe (target size ∼200kb; b, base) and the Y-specific probe (target size &gt;2Mb), demonstrate that the binding sites of the two probes can be distinguished in the same nucleus on the basis of domain size.
A somatic cell-derived system for studying both early and late mitotic events in vitro
Authors: Nakagawa Junichi , Kitten Gregory T. , Nigg Erich A. 
Published: April 2021

ABSTRACT
We describe a cell-free system for studying mitotic reorganization of nuclear structure. The system utilizes soluble extracts prepared from metaphase-arrested somatic chicken cells and supports both the disassembly and subsequent partial reassembly of exogenous nuclei. By fluorescence microscopy, biochemical fractionation, protein phosphorylation assays and electron microscopy, we show that chicken embryonic nuclei incubated in extracts prepared from metaphase-arrested chicken hepatoma cells undergo nuclear envelope breakdown, lamina depolymerization and chromatin condensation. These prophase-like events are strictly dependent on ATP and do not occur when nuclei are incubated in interphase extracts. Compared to interphase extracts, metaphase extracts show increased kinase activities toward a number of nuclear protein substrates, including lamins and histone Hl ; moreover, they specifically contain four soluble phosphoproteins of Mr 38 000, 75 000, 95 000 and 165 000. Following disassembly of exogeneous nuclei in metaphase extracts, telophase-like reassembly of a nuclear lamina and re-formation of nuclear membranes around condensed chromatin can be induced by depletion of ATP from the extract. We anticipate that this reversible cell-free system will contribute to the identification and characterization of factors involved in regulatory and mechanistic aspects of mitosis.
Persistence of major nuclear envelope antigens in an envelope-like structure during mitosis in Drosophila melanogaster embryos
Authors: Harel Amnon , Zlotkin Efrat , Nainudel-Epszteyn Sandra , Feinstein Naomi , Fisher Paul A. , Gruenbaum Yosef 
Published: April 2021

ABSTRACT
Using monoclonal antibodies, we followed the fate of three different nuclear envelope proteins during mitosis in Drosophila early embryos by indirect immunofluorescence microscopy. Two of these proteins, lamin and otefin, a newly characterized nuclear envelope polypeptide with an apparent Mr of 53 000, are apparently present in an envelope-like structure that is present throughout mitosis. Immunoelectron microscopy of interphase nuclei indicates that otefin, like lamin, is not a component of nuclear pore complexes. In contrast with lamin and otefin, gpl88, a putative pore complex component, was completely redistributed through the surrounding cytoplasm during prophase in comparable early embryo specimens and was present in an envelope only in interphase. Together with previous morphological studies by other workers, these data suggest that the entire mitotic apparatus including condensed chromosomes and spindle is enclosed by an envelope throughout mitosis during early embryo-genesis in Drosophila. This ‘spindle envelope’, as it has been named by others, contains both lamin and otefin but probably not pore complex proteins.
A new monoclonal antibody to a cell-surface antigen that distinguishes luminal epithelial and myoepithelial cells in the rat mammary gland
Authors: Mahendran R. S. , O’Hare M. J. , Ormerod M. G. , Edwards P. A. W. , McIlhinney R. A. J. , Gusterson B. A. 
Published: April 2021

ABSTRACT
A monoclonal antibody (25.5) has been produced that recognises luminal epithelial cells of the rat mammary gland. This antibody together with monoclonal anti-CALLA antibodies, which react with mammary myoepithelial cells, has been used in biochemical, immunocytochemical and flow cytometric studies. Antibody 25.5 bound to proteins of molecular weight 70K and 25K (K=103Mr) in both the rat milk fat globule membrane and in single cell suspensions prepared from the virgin adult rat mammary gland. Anti-CALLA antibody (J5), recognised a 93-100K protein in the gland extracts, which co-electrophoresed with the CALLA/CD-10 antigen from NALM-6 acute lymphoblastic leukaemia cell line.
Antibody 25.5 bound to the luminal surface of rat mammary epithelial cells at all stages of development from neonatal through to pregnancy, lactation and involution. CALLA immunoreactive staining has previously been shown on basally located pre-sumptive myoepithelial cells at all stages of development. Flow cytometric analyses demonstrated that 25.5 and anti-CALLA antibodies stained independent cell populations in suspensions of single cells prepared from purified epithelial elements from the mammary gland of adult virgin rat.
The rna content of the nucleolus and nucleolus-like inclusions in the anther of Lilium estimated by an improved rnase-gold labelling method
Authors: Sato S. , Willson C. , Dickinson H. G. 
Published: April 2021

ABSTRACT
Using RNase-gold labelling a comparative estimation has been made of the RNA content of the nucleofi and nucleofus-like inclusions in anther cells of Lilium henryi Thunb. Pretreatment with glow electric discharge prior to application of the RNase-gold labelling remarkably lowered the level of nonspecific adsorption and allowed specific labelling of RNA-containing structures. In the tap-etal cells, the nucleofi, the perichromatin material and the dense bodies labelled heavily, but both the interchromatin region and the condensed chromatin did not bind the label. The nucleofus-like inclusions, which were found in the nucleoplasm of meiotic cells at the tetrad stage, and were termed‘nuclear nucleofus-like bodies (NLBs)’ in the present study, also showed a high response compared with both the loosened and the condensed chromatin; this labelling was some 60% of that observed over the nucleofi of somatic cells. Another type of nuclear NLB was differentiated from the predominant type of NLB by a conspicuous electron opacity, reduced size, and strong labelling with the RNase-gold complex, suggesting that some nuclear NLBs may be highly condensed. The labelling over the cytoplasmic nucleofoids (nucleofus-like inclusions found in the cytoplasm) was only 50 % of that over the nuclear NLBs, although both are similar in ultrastructural texture.
Transformed sweat gland and nasal epithelial cell lines from control and cystic fibrosis individuals
Authors: Buchanan Janet A. , Yeger Herman , Tabcharani Joseph A. , Jensen Timothy J. , Auerbach Wojtek , Hanrahan John W. , Riodan John R. , Buchwald Manuel 
Published: April 2021

ABSTRACT
We undertook to extend the in vitro lifespan of epithelial cell cultures useful for the study of the cellular defect underlying cystic fibrosis (CF). Primary cultures from sweat glands of four CF and four non-CF and from nasal polyps of one non-CF and two CF individuals were transformed using a chimaeric virus, Ad5/SV40 1613 ori−. The extended lifespans ranged from 20 to more than 250 population doublings beyond that of the primary cultures. Despite some degree of aneuploidy (as assayed by total cellular DNA content) all samples tested retained at least one copy of the region of chromosome 7 containing the CF gene (as assayed by probing with flanking DNA markers). Epithelial characteristics, including an epithelioid morphology, tight junctions and desmosomes, apical microvilli, keratin networks, and dome formation were positive in the majority of cells examined, although variably expressed. All cells tested demonstrated outwardly rectifying chloride channels by patch clamp, with some from non-CF cells responsive to the catalytic subunit of cyclic AMPdependent protein kinase. The cells were used for DNA transfection assays with selectable marker genes in appropriate vectors, in order to develop methodology for assaying the function of the CF gene product and the effects of mutations.
Interphase microtubule dynamics are cell type-specific
Authors: Wadsworth P. , Mcgrail M. 
Published: April 2021

ABSTRACT
The rate and pattern of microtubule polymer loss in interphase cells have been examined using nocod-azole to block microtubule assembly. Cells were incubated with high concentrations of nocodazole for various times and the pattern of microtubule disassembly was determined using tubulin immunofluorescence. Polymer loss was quantitated by measuring the decrease in percentage of cell area occupied by microtubules. The results demonstrate that microtubules in diverse cells disassemble individually and asynchronously. In addition, these quantitative measurements reveal that epithelial and fibroblast cells display strikingly different kinetics of polymer loss. In fibroblasts, polymer loss is rapid, with a half-time of 4 min at 37°C. In epithelial cells, loss of 60% of the microtubules occurs with a half-time of 18 min; the remaining 40% of the microtubules disassemble much more slowly (average half-time of 72 min). To demonstrate that these differences were not due to species differences among various cells assayed in these experiments, epithelial and fibroblast cells derived from primary cultures of newt lung have been examined. Again, fibroblast and epithelial cell microtubule dynamics could be readily distinguished. To determine if modifications to epithelial cell microtubules contribute to their stability, microtubules were completely disassembled and allowed to regrow. The rate of polymer loss for recently regrown microtubules was more rapid than microtubules in control cells, indicating that stability increases with time after assembly.
Phosphoproteins associated with cyclic nucleotide stimulation of ciliary motility in Paramecium
Authors: Bonini Nancy M. , Nelson David L. 
Published: April 2021

ABSTRACT
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 (&gt;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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACTIn 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.