bluuebunny/crossref_metadata_2025_split huggingface dataset:
Amplification of the Golgi complex in MDCK cells secreting human growth hormone
Authors: Rudick Victoria L. , Rudick Michael J. , Brun-Zinkernagel Anne-Marie 
Published: April 2021

ABSTRACT
MDCK cells were transfected with pXGH5, a plasmid containing the human growth hormone (hGH) gene, and permanently expressing cell lines were selected. Clone 3A cells, which secrete quantities of hGH through both apical and basolateral surfaces, were examined in detail. Immunofluorescence analysis using anti-hGH antibody revealed bright perinuclear staining coinciding with the area delineated by anti-p52 kDa protein (a resident Golgi protein) antibody. There appeared to be less Golgi-specific fluorescence in untransfected cells. This difference correlated with an increased amount of 52 kDa in the clone 3A cells. Morphometric analysis was performed on electron micrographs of clone 3A and untransfected cells using the fractionator to estimate average number of Golgi stacks per cell, and values were statistically analyzed. It was found that clone 3A cells contained 3.3 and untransfected cells 1.6 stacks (P ≤0.005), respectively. When clone 3A cells were placed into defined medium, the synthesis and secretion of hGH declined 4-fold, and the number of Golgi stacks also decreased to the untransfected level within seven days. The number of Golgi stacks per untransfected cell was not affected by the presence of exogenous hGH, indicating that Golgi amplification was directly related to secretory demand. Generation times and cell volumes were identical for both cell types under all growth conditions. In addition, the kinetics of protein secretion from radiolabelled cells demonstrated that clone 3A cells generally secrete lower amounts of endogenously synthesized apical proteins than do untransfected cells, while basolateral secretion remains the same. In both cases hGH comprised only about 10% of total secretory proteins, so that the increase in total protein secretion did not seem to warrant the two-fold elaboration of Golgi by 3A cells. But there might be significant amounts of hGH which traverse the Golgi to end up in lysosomes, rather than being secreted, leading to Golgi amplification.
Human cyclin D1 encodes a labile nuclear protein whose synthesis is directly induced by growth factors and suppressed by cyclic AMP
Authors: Sewing Andreas , Bürger Christiane , Brüsselbach Sabine , Schalk Christian , Lucibello Frances C. , Müller Rolf 
Published: April 2021

ABSTRACT
We show that the cyclin D1 gene is regulated by a variety of growth factors in human diploid fibroblasts (WI- 38). Expression of cyclin D1 mRNA is low in quiescent WI-38 cells and reaches a maximum around 10 hours after serum stimulation, i.e. approximately 8 hours prior to the onset of DNA synthesis. A cyclin D1-specific anti- serum raised against a bacterially expressed fusion pro- tein detected a 39 kDa polypeptide in WI-38 cells. In agreement with the RNA expression data, cyclin D1 pro- tein synthesis is also serum-inducible, reaching a maxi-mum around 9 hours post-stimulation. The results obtained by pulse-chase experiments, cell fractionation and immunostaining techniques strongly suggest that cyclin D1 is a labile protein (t½ ≈ 38 min), which is located in the nucleus. Cyclin D1 is directly induced by growth factors, i.e. in the presence of cycloheximide, and its expression does not significantly fluctuate during the cell cycle in synchronized cells. Cyclin D1 therefore fun-damentally differs from “classical” cyclins, such as the mitotic cyclin B, whose expression is clearly cell cycle-dependent. Cyclin D1 may rather establish a direct link between growth control mechanisms and the cell cycle. Interestingly, cyclin D1 expression is stimulated by the protein kinase C activator TPA, but suppressed by dibu-tyryl-cAMP and the adenylate cyclase inducer forskolin, pointing to multiple regulatory pathways controlling cyclin D1 expression.
Abnormal anaphase resolution (aar): a locus required for progression through mitosis in Drosophila
Authors: Gomes R. , Karess R. E. , Ohkura H. , Glover D. M. , Sunkel C. E. 
Published: April 2021

ABSTRACT
We describe a new mitotic locus of Drosophila melanogaster required for the progression through mitosis in the syncytial embryo and in late larval development. The locus aar (abnormal anaphase resolution) maps to the cytological interval 85E7-F16 and was identified by two alleles. The aar1 allele causes pupal lethality. Larval neuroblasts show an elevated mitotic index with high chromosome condensation and stretched and lagging chromatids during anaphase. aar2 produces fully viable but sterile females. aar1/aar2 females lay eggs that develop mitotic figures with similar abnormalities to those observed in neuroblasts. Indirect immunofluorescence of these embryos indicates that the centrosome cycle appears normal, although some abnormal spindle microtubules can be seen during mitosis.
Local opening of the DNA double helix in eukaryotic cells detected by osmium probe and adduct-specific immunofluorescence
Authors: Paleček Emil , Robert-Nicoud Michel , Jovin Thomas M. 
Published: April 2021

ABSTRACT
The structure of DNA in mouse fibroblast 3T3 cells has been investigated with the single-strand-selective probe, the complex of osmium tetroxide and 2,2 -bipyridine (Os,bipy). DNA-Os,bipy adducts in the cells were detected by immunofluorescence using a highly specific, affinity-purified polyclonal antibody. Treatment of living cells with the chemical probe led to a distinct but nonuniform nuclear staining. We attribute the positive nuclear staining to the existence of single-stranded and distorted DNA regions in the living cell. Confocal laser scanning microscopy revealed dark areas corresponding to nucleoli and regions of condensed chromatin. These conclusions were supported by the results of experiments in which the chemical probe was applied to fixed cells treated with 45% acetic acid or with acidic buffers (pH 1.8-2.5). An inverse staining pattern was obtained, characterized by intense immunofluorescence of the condensed chromatin regions. Thus, the structural transitions and/or chemical alterations (e.g. depurination) induced by acid treatment increase the accessibility of normally unreactive DNA bases. We conclude that open DNA structures recognized by the chemical probe in the cells prior to their fixation are contained mainly in decondensed and transcriptionally active chromatin, but are virtually absent from nucleoli and condensed chromatin.
Establishment of renal proximal tubule cell lines by targeted oncogenesis in transgenic mice using the L-pyruvate kinase-SV40 (T) antigen hybrid gene
Authors: Cartier N. , Lacave R. , Vallet V. , Hagege J. , Hellio R. , Robine S. , Pringault E. , Cluzeaud F. , Briand P. , Kahn A. , Vandewalle A. 
Published: April 2021

ABSTRACT
Targeted oncogenesis allowed us to obtain two cell lines which have been derived from the proximal tubule of kidney from transgenic mice harbouring the simian virus (SV40) large T and small t antigens placed under the control of the 5 regulatory sequence from the rat L-type pyruvate kinase (L-PK) gene. The cell lines (PKSV-PCT and PKSV-PR cells) were derived from early (PCT) and late (Pars Recta, PR) microdissected proximal tubules grown in D-glucose-enriched medium. In such conditions of culture, both cell lines exhibited L-PK transcripts, a stable expression of SV40-encoded nuclear large T antigen, a prolonged life span but failed to induce tumors when injected sub-cutaneously into athymic (nu-nu) mice. Confluent cells, grown on plastic support or porous filters, were organized as monolayers of polarized cuboid cells with well developed apical microvilli and formed domes. Both cell lines exhibited morphological features of proximal tubule cells with villin located in the apical brush-border and subtantial amounts of hydrolase activity. By immunofluorescence studies using specific antibodies, aminopeptidase N appeared restricted to the apical microvillar domain, whereas the H2 histocompatibility antigen was distributed in the cytoplasm and lateral membranes. These results demonstrate that the proximal morphological phenotype has been fully preserved in these cultured cells derived from tissue-specific targeted oncogenesis in transgenic mice.
Effect of monensin on plant Golgi: re-examination of the monensin-induced changes in cisternal architecture and functional activities of the Golgi apparatus of sycamore suspension-cultured cells
Authors: Zhang Guo Feng , Driouich Azeddine , Staehelin L. Andrew 
Published: April 2021

ABSTRACT
We have re-examined the effects of the ionophore monensin on the Golgi apparatus of sycamore maple suspension-cultured cells using a combination of high pressure freezing, immunocytochemical and biochemical techniques. Exposure of the cells to 10 μ M monensin, which reduces protein secretion by ∼90%, resulted first in the swelling of the trans-Golgi network, then of the trans-most trans-cisterna, the remaining trans-cisternae, and finally of the cis and medial cisternae. We postulate that these different rates of swelling reflect an underlying hierarchy of compartmental acidification with the trans-Golgi network being the most acidic compartment. Recovery occurred in the reverse sequence. Previous studies have suggested that the large swollen vesicles that accumulate in the cytoplasm of monensintreated cells arise from the swelling and detachment of entire trans-cisternae. However, based on the many membrane blebbing configurations seen in association with the trans-Golgi network and the trans-Golgi cisternae of monensin-treated cells, and the fact that the surface area of the trans-Golgi cisternae is about five times greater than the surface area of the swollen vesicles, it appears that the swollen vesicles are produced by a budding mechanism. After 35-40 min of monensin treatment, cells with smaller, non-swollen, compact Golgi stacks began to appear and rapidly increased in number, contributing &gt;60% of the cell population after 60 min and &gt;80% after 100 min. In contrast, large numbers of swollen vesicles persisted in the cytoplasm of all cells for over 100 min. Since azide treatment of monensin-treated cells can prematurely induce the unswelling response and cellular ATP levels drop substantially after 45 min of monensin treatment, we propose that un-swelling of the Golgi stacks is due to a monensin-induced decline in ATP levels in the cells.
Immunocytochemical labeling of the high pressure frozen cells with anti-xyloglucan antibodies demonstrated that the concentration of xyloglucan, a hemicellulose, in the swollen vesicles increased with time. This increase in vesicle contents may explain why these swollen vesicles do not contract in parallel with the Golgi stacks. In vivo labeling experiments with [3H]fucose, [3H]UDP-glucose and [3H]leucine demonstrated that monensin-treatment not only inhibited protein secretion, but also cellulose synthesis. Protein synthesis, on the other hand, was reduced only slightly during the first 30 min of treatment, but quite strongly between 30 and 60 min, consistent with the observed drop in ATP levels after &gt;40 min of exposure to monensin. Taken together, our findings indicate that the Golgi apparatus of plant cells responds to monensin in a much more differentiated manner than envisaged to date, thereby opening up interesting new possibilities for analyzing its functional organization.
Epithelial sphingolipid sorting is insensitive to reorganization of the Golgi by nocodazole, but is abolished by monensin in MDCK cells and by brefeldin A in Caco-2 cells
Authors: Meer Gerrit van , ’t Hof Wouter van 
Published: April 2021

ABSTRACT
In epithelial MDCK and Caco-2 cells, short-chain analogs of glucosylceramide and sphingomyelin are delivered from the Golgi to the cell surface with different apical/basolateral polarities, which results in an apical enrichment of the glycolipid glucosylceramide over the phospholipid sphingomyelin. Here, we have interfered with the integrity of the Golgi complex in various ways and tested the effects on lipid transport and sorting. Nocodazole, which depolymerizes microtubules, dispersed the Golgi over the cytoplasm of MDCK cells and reduced transport of newly synthesized C6-NBD-(N-6[7-nitro-2,1,3-benzoxadiazol-4-yl]aminocaproyl)-glucosylceramide and C6-NBD-sphingomyelin to the apical surface by 40%. The lipids were not mistargeted to the basolateral surface and upon removal of nocodazole, apical transport recovered. Nocodazole did not affect the apical enrichment of glucosylceramide over sphingomyelin. The ionophore monensin led to swelling of the Golgi of MDCK cells and inhibited lipid transport to the cell surface by 30-50%. Whereas sphingomyelin transport to both surface domains was equally affected, monensin mainly inhibited apical transport of glucosylceramide. At 10-20 μM of monensin, the two lipids displayed the same polarity of delivery: sorting between the two lipids was abolished. Brefeldin A at 1 μg/ml, which resulted in disruption of the Golgi in HepG2 cells and completely inhibited protein secretion, had no inhibitory effect on transport of the C6-NBD-lipids to the surface. The same was observed in Caco-2 cells. However, brefeldin A selectively shifted transport of sphingomyelin towards the apical direction which abolished the apical enrichment of glucosylceramide over sphingomyelin. Caco-2 cells were used because in MDCK cells brefeldin A did not change Golgi structure nor lipid transport and sorting. In summary, modification of the Golgi by monensin and brefeldin A, but not nocodazole, interfered with the sorting event by which glucosylceramide is enriched over sphingomyelin in the transport pathway from the Golgi to the apical surface.
Different patterns of rDNA distribution in Pisum sativum nucleoli correlate with different levels of nucleolar activity
Authors: Highett Martin I. , Rawlins David J. , Shaw Peter J. 
Published: April 2021

ABSTRACT
We have used in situ hybridization with probes to rDNA, labelled either with digoxygenin or directly with fluorescein, to determine the arrangement of these genes within the nucleoli of Pisum sativum L. root cells. Confocal laser scanning microscopy was used to image the three-dimensional structures revealed, but we have also compared this technique with deconvolution of conventional (wide-field) fluorescence images measured with a cooled CCD camera, and have shown that the results are remarkably similar. When the deconvolution technique was applied to the confocal data it gave clearer images than could be achieved by confocal microscopy alone. We have analysed the distribution of rDNA in the different cell types observable in root tips: the quiescent centre; active meristematic cells; and relatively differentiated root cap, epidermal and cortical cells. In addi-tion to four perinucleolar knobs of condensed, inactive rDNA genes, corresponding to the four nucleolar organizers in P. sativum, which were the most brightly labelled structures, several characteristic patterns of intranucleolar labelling were apparent, including bright foci, large central chromatin masses, and fine, decondensed interconnecting fibres. The larger and more active the nucleolus, the smaller the proportion of condensed perinucleolar rDNA. In some large and active meristematic nucleoli, all the internal rDNA is decondensed, showing that transcription cannot be restricted to the bright foci, and is most likely to occur on the decondensed fibres.
Degradation of an extracellular matrix: sea urchin hatching enzyme removes cortical granule-derived proteins from the fertilization envelope
Authors: Mozingo Nancy M. , Hollar Lisa R. , Chandler Douglas E. 
Published: April 2021

ABSTRACT
The sea urchin fertilization envelope is an extracellular matrix assembled at fertilization to prevent polyspermy and protect the embryo during early development. During hatching, the embryo secretes a proteolytic hatching enzyme which dissolves the fertilization envelope, allowing a ciliated blastula to swim free. In this study we examined ultrastructural changes in the fertilization envelope during degradation of this matrix by hatching enzyme. The completed fertilization envelope is a trilaminar structure consisting of a dense, central layer of filaments sandwiched between surface coats of paracrystalline material. Hatching enzyme disassembles this matrix by degrading the paracrystalline layers and removing macromolecules from the central layer leaving behind a thin matrix of loosely woven fibers.
Identification of macrophages and dendritic cells in the osteopetrotic (op/op) mouse
Authors: Witmer-Pack Margit D. , Hughes Derralynn A. , Schuler Gerold , Lawson Linda , McWilliam Andrew , Inaba Kayo , Steinman Ralph M. , Gordon Siamon 
Published: April 2021

ABSTRACT
We used a panel of monoclonal antibodies and immuno-cytochemistry to identify macrophages and dendritic cells in mice that are deficient in macrophage colony stimulating factor (M-CSF or CSF-1) because of the recessive osteopetrotic (op/op) mutation. Prior work had shown that osteopetrosis is associated with a lack of osteoclasts, phagocytic cells required for remodelling in bone. Additional macrophage populations proved to be very M-CSF dependent. op/op mice had few and some-times no peritoneal cavity phagocytes, splenic marginal zone metallophils, and lymph node subcapsular sinus macrophages. Other populations, however, reached sub-stantial levels in the absence of M-CSF, including phago-cytes in the thymic cortex, splenic red pulp, lymph node medulla, intestinal lamina propria, liver (Kupffer cells), lung (alveolar macrophages) and brain (microglia). Dendritic cells, which are specialized accessory cells for T-dependent immune responses and tolerance, were read-ily identified in skin and in the T-dependent regions of spleen, lymph node and Peyer’s patch. The identifica-tion of dendritic cells utilized antibodies to MHC class II products and four different antigens that are pri-marily expressed by these accessory cells. Our findings indicate that only a few macrophage populations are critically dependent upon M-CSF in vivo. With respect to dendritic cells, the data are consistent with prior in vitro work where it was noted that GM-CSF but not M-CSF supported dendritic cell viability, function and growth.
Consequences of topoisomerase II inhibition in early embryogenesis of Drosophila revealed by in vivo confocal laser scanning microscopy
Authors: Buchenau Peter , Saumweber Harald , Arndt-Jovin Donna J. 
Published: April 2021

ABSTRACT
The regulation of DNA topology by topoisomerase II from Drosophila melanogaster has been studied extensively by biochemical methods but little is known about its roles in vivo. We have performed experiments on the inhibition of topoisomerase II in living Drosophila blastoderm embryos. We show that the enzymatic activity can be specifically disrupted by microinjection of antitopoisomerase II antibodies as well as the epipodophyllotoxin VM26, a known inhibitor of topoisomerase II in vitro. By labeling the chromatin of live embryos with tetramethylrhodamine-coupled histones, the effects of inhibition on nuclear morphology and behaviour was followed in vivo using confocal laser scanning microscopy. Both the antibodies and the drug prevented or hindered the segregation of chromatin daughter sets at the anaphase stage of mitosis. In addition, high concentrations of inhibitor interfered with the condensation of chromatin and its proper arrangement into the metaphase plate. The observed effects yielded non-functional nuclei, which were drawn into the inner yolk mass of the embryo. Concurrently, undamaged nuclei surrounding the affected region underwent compensatory division, leading to the restoration of the nuclear population, and thereby demonstrating the regulative capacity of Drosophila blastoderm embryos.
Astral and spindle forces in PtK2 cells during anaphase B: a laser microbeam study
Authors: Aist James R. , Liang Hong , Berns Michael W. 
Published: April 2021

ABSTRACT
Rat kangaroo kidney epithelium (PtK2) cells develop prominent asters and spindles during anaphase B of mitosis. It has been shown that severing the spindle at early anaphase B in living PtK1 cells results in a dramatic increase in the rate of pole-pole separation. This result suggested that the asters pull on the spindle poles, putting tension on the spindle, while the spindle acts as a governor, limiting the rate of pole separation. To further test these inferences, we used a UV-laser microbeam to damage one of the two asters in living PtK2 cells at early anaphase B and monitored the effects on individual spindle pole movements, pole-pole separation rates and astral microtubules (MTs). Irradiation at the estimated position of a centrosome greatly reduced its array of astral MTs and nearly stopped the movement of the irradiated pole, whereas the sister pole retained its normal array of astral MTs and actually accelerated. Control irradiations, either close to the estimated position of the centrosome or beside the spindle at the equator, had little or no effect on either spindle pole movements or astral MTs. These results support the inferences that during anaphase B in living PtK cells, the central spindle is under tension generated by pulling forces in the asters (presumably MT-mediated) and that the spindle generates counterforces that limit the rate of pole separation. The results also suggest that the central spindle in living PtK cells may be able to generate a pushing force.
Signal transduction and nuclear targeting: regulation of transcription factor activity by subcellular localisation
Authors: Whiteside Simon T. , Goodbourn Stephen 
Published: April 2021

ABSTRACT
Over the last few years a number of international meetings have boasted titles along the lines of “from receptor to nucleus” reflecting a desire to construct a pathway linking the endpoints of different regulatory events. However, for historical and technical reasons, research effort has been focused overwhelmingly on receptor/ligand or transcription factor/DNA interactions. The task confronting biologists now is to link up these two extremes. Our present under-standing of these intermediary processes is in many cases rudimentary, but it is clear that signal transduction from membrane to nucleus often involves many steps, and it also appears that several pathways can use common intermediates, which raises interesting questions in speed and specificity.