bluuebunny/crossref_metadata_2025 huggingface dataset:
Organization and characterization of fibrillar collagens in fish scales in situ and in vitro
Authors: Zylberberg L. , Bonaventure J. , Cohen-Solal L. , Hartmann D. J. , Bereiter-Hahn J.
Published: April 2021

The characterization of the fibrillar collagens and the cellular control of their spatial deposition were studied in fish scales using immunofluorescence, electron microscopy, electrophoretic and HPLC analyses, immunoprecipitation and hybridization with cDNA probes. This study was carried out on undisturbed and regenerating scales in situ and in organ and cell cultures from regenerating scales. The hyposquamal scleroblasts forming a pseudoepithelium show an apico-basal polarization and synthesize thick collagen fibrils (100 nm) organized in a plywood pattern as long as the integrity of the cell-cell and cell-collagenous matrix contacts are preserved. In culture, scleroblasts become fibroblast-like and produce an unordered meshwork of thin collagen fibrils (30 nm). Comparison of the synthesized collagens in culture with those extracted from the scales indicates that culture conditions modify fibrillogenesis but do not change the expression of fibrillar collagen genes. Type I collagen, the prédominent component, is associated with the minor type V collagen. Type HI collagen was not present. In type I collagen, a third chain, α3 chain, was identified. The ratio between the 3 chains suggests the coexistence of two heterotrimers (αl(I))2 α2(1) and αl(I) α2(1) α3(I). Analysis by HPLC and electrophoresis of the cyanogen bromide-derived peptides obtained from the purified α3 chain support the hypothesis that αl(I) and α3(I) chains are encoded by two different genes. The presence of the two types of heterotrimers in vivo as well as in vitro could correspond to an innate property of the goldfish scleroblasts. Despite the fact that teleost cyanogen bromide-derived peptides differ from those of higher vertebrates, homologies with the mammalian collagen genes (human, for example) are sufficient to allow the detection of mRNA transcripts for al(I), α2(I) and α2(V) from confluent scleroblast cultures with human probes.
Examination of transcellular membrane protein polarity of bovine aortic endothelial cells in vitro using the cationic colloidal silica microbead membrane-isolation procedure
Authors: Stolz Donna Beer , Jacobsonf Bruce S.
Published: April 2021

In this report we describe a rapid, high-yield protocol for the isolation of apical (AP) and basolateral (BL) plasma membrane domains from monolayers of bovine aortic endothelial cells (BAECs) grown on tissue culture dishes as well as microcarrier beads. Using a modified cationic colloidal silica microbead membrane-isolation procedure, which deposits a uniform silica-polyacrylate pellicle over the entire AP membrane surface, a 4- to 9.6-fold relative enrichment of AP membrane and a 3.55- to 3.67-fold relative enrichment of BL membrane was obtained when the isolated domains were examined for silica and Na+/K+-ATPase, respectively. Immunoblotting of the isolated membrane domains displayed the presence of angiotensin-converting enzyme (ACE) exclusively in the AP domain and collagen receptors (CRs) highly enriched in the BL membrane domain when monolayers were grown on a gelatin substratum.
The Drosophila ncd microtubule motor protein is spindle-associated in meiotic and mitotic cells
Authors: Hatsumi Machiko , Endow Sharyn A.
Published: April 2021

The nonclaret disjunctional (ncd) protein is required for normal chromosome distribution in oocytes and early embryos. Mutants of ncd cause frequent nondisjunction and loss of chromosomes, suggesting a role for the protein in spindle function or chromosome movement in meiosis and early mitosis. The ncd protein contains a region of predicted sequence similarity to the microtubule motor protein, kinesin. In vitro motility assays have demonstrated that ncd is a motor that unexpectedly moves toward the minus ends of microtubules, opposite to the direction of kinesin movement. Using antibodies directed against nonconserved regions of the protein, we have localized the ncd motor protein to the meiotic and early mitotic spindle, and to spindles in a mitotically dividing cultured cell line. Its presence in the spindle of meiotic and mitotic cells implies a role for the protein as a spindle motor. The motor may play an essential role in establishing spindle bipolarity in meiosis.
Characterization and immunolocalization of RNA polymerase I transcription factor UBF with anti-NOR serum in protozoa, higher plant and vertebrate cells
Authors: Rodrigo Rosa M. , Rendón M. Carmen , Torreblanca José , García-Herdugo Gregorio , Moreno Francisco J.
Published: April 2021

We have used anti-NOR serum from a patient with rheumatoid arthritis, to study its reactivity on different phylogenetically separated species such as protozoa, higher plants, birds and mammals. The biochemical characteristics of the antigens detected after applying monoand two-dimensional electrophoresis and electrophoretic transfers confirm that they correspond to the rRNA polymerase I transcription factor UBF. We have demonstrated the different molecular sizes, depending on the cell complexity, but the same neutral isoelectric points in whole cell extracts of the different species. We have also demonstrated an immunolocalization of this transcription factor to the fibrillar component in all the species studied. These results suggest a high conservation of UBF throughout evolution and the possibility of using this anti-NOR serum as a tool for the study of the structure, nucleolar organization and functional roles of the different nucleolar components.
Human osteoblasts in culture synthesize collagenase and other matrix metalloproteinases in response to osteotropic hormones and cytokines
Authors: Meikle Murray C. , Bord Sharyn , Hembry Rosalind M. , Compston Juliet , Croucher Peter I. , Reynolds John J.
Published: April 2021

Collagenase production by rodent osteoblasts in response to calciotropic hormones has led to the hypothesis that bone cells play a major role in bone resorption by degrading the surface osteoid layer, thereby exposing the underlying mineralized matrix to osteoclastic action. Many studies suggest, however, that this model might not apply to bone resorption in the human. Human osteoblasts have been shown to produce gelatinase-A (72 kDa) and TIMP-1 (tissue inhibitor of metalloproteinases), but previous investigators have been unable to demonstrate the synthesis of collagenase by human osteoblasts either constitutively or in response to bone resorptive agents.

In the present study the ability of human osteoblasts to produce the matrix metalloproteinases (MMPs) collagenase, gelatinase and stromelysin, and their specific inhibitors TIMPs-1 and 2, was examined using highly sensitive and specific antisera and by zymography. Semi-quantitative histomorphometric data showed that cells cultured on either glass or a type I collagen substratum constitutively synthesized gelatinase-A and TIMP-1. On type I collagen, however, a small proportion of unstimulated cells produce both collagenase (7%) and gelatinase-B (95 kDa; 3%). Treatment of cells with either parathyroid hormone (PTH), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), or partially purified mononuclear cell conditioned medium (MCM), stimulated the synthesis of collagenase, gelatinase-B and stromelysin; MCM was 2-to 3-fold more potent than either PTH or 1,25(OH)2D3.

Zymography using SDS/PAGE on conditioned media from cells cultured on type I collagen films revealed the presence of active gelatinase-A and that MCM stimulated progelatinase-B synthesis. Inhibitor gel zymography confirmed that TIMP-1 was constitutively synthesized and that its expression was increased by MCM treatment; TIMP-2 could not be detected.

These data confirm the findings of previous investigators that human osteoblasts constitutively produce gelatinase-A and TIMP-1, and show for the first time that they can also be stimulated to synthesize collagenase, gelatinase-B and stromelysin. The working hypothesis that osteoblast MMPs play an important role in bone resorption would therefore appear to be as valid for human as it is for rodent bone.
Human articular surface chondrocytes initiate alkaline phosphatase and type X collagen synthesis in suspension culture
Authors: Stephens Myra , Kwan Alvin P. L. , Bayliss Michael T. , Archer Charles W.
Published: April 2021

The type X collagen is a short chain collagen associated with calcific cartilage and/or the expression of the hypertrophic chondrocyte phenotype. In articular cartilage, type X collagen is restricted to the basal zone of calcified cartilage adjacent to the subchondral bone. However, during pathological change such as in osteoarthritis, the synthesis of type X collagen becomes more widespread but never extends to the articular surface. Using immunocytochemistry and fluorography of newly synthesised collagens, we report that surface articular chondrocytes (which occupy the uppermost 10– 15% of the tissue depth) from normal human cartilage initiate de novo synthesis of both type X collagen and alkaline phosphatase when maintained in suspension culture.
Disorganization of the Golgi complex and the cytoplasmic microtubule system in CHO cells exposed to okadaic acid
Authors: Thyberg Johan , Moskalewski Stanislaw
Published: April 2021

A combination of immunocytochemical and electron microscopic methods was used to study the effects of okadaic acid, a specific inhibitor of protein phosphatase types 1 and 2A, on the Golgi complex and the microtubule system of interphase CHO cells. At a concentration of 0.25 M and within 2-3 h of exposure, okadaic acid caused a reversible disorganization of the Golgi complex, observed as a disintegration of the stacks of cisternae and formation of clusters of tubules and vesicles dispersed in the cytoplasm. At the same time, staining for mannosidase II was shifted from the Golgi stacks to the endoplasmic reticulum, whereas the clusters of tubules and vesicles for the main part were negative. This change in localization of the enzyme was not blocked by cycloheximide and thus not dependent on ongoing protein synthesis. The changes in the morphology of the Golgi complex were coordinated in time with a remodelling of the microtubule system, observed as a reduction in the number of microtubules, a tendency of the remaining microtubules to arrange in an aster-like pattern, and an increased sensitivity to low concentrations of the microtubule-disruptive drug nocodazole. After removal of the drug, the microtubule system was rapidly normalized (1-2 h) and subsequently also the Golgi complex (4-8 h). The results suggest that okadaic acid induces a redistribution of the Golgi stacks into the endoplasmic reticulum, leaving the trans-most elements behind as tubules and vesicles. Altogether, the changes in the organization of the Golgi complex and the microtubule system caused by okadaic acid resemble the changes previously described to occur in these organelles at the onset of mitosis, possibly indicating a similar mechanism of action involving specific protein phosphorylation.
Intracellular trafficking and the parasitophorous vacuole of Leishmania mexicana-infected macrophages
Authors: Russell David G. , Xu Songmei , Chakraborty Prasanta
Published: April 2021

The continued success of Leishmania as an intra-macrophage parasite is dependent on its ability to survive within an acidic intracellular compartment, resist degradation by lysosomal hydrolases, exploit the host cell as a source of nutrients, and avoid the macrophage’s antigen-presenting capabilities. All these requirements are dependent on the properties of the parasitophorous vacuole in which Leishmania resides. This study shows that the vacuole possesses membrane proteins characteristic of a lysosome, and has MHC class II molecules. The trafficking of a variety of endocytic markers supports this finding. However, a temporal study up to 14 days post-infection indicates that, as it matures, the vacuole gains mannose 6-phosphate receptor, and becomes more accessible to endocytosed ligand, suggesting that the vacuole has functionally translocated from a lysosomal to late endosomal compartment. Endocytosed material was detected in the flagellar pocket and inside the amastigote, demonstrating parasite uptake of intra-vacuolar material. Careful analysis of amastigotes suggests that they avoid antigen presentation by their host cell by limiting the release of potential antigens. These findings significantly extend our understanding of the mechanisms employed by Leishmania to ensure its survival in the macrophage.
N-cadherin and N-CAM in myoblast fusion: compared localisation and effect of blockade by peptides and antibodies
Authors: Mege R. M. , Goudou D. , Diaz C. , Nicolet M. , Garcia L. , Geraud G. , Rieger F.
Published: April 2021

The expression and distribution of two cell adhesion molecules, N-cadherin and N-CAM, at the surface of cultured leg muscle cells from 11-day-old chicken embryos were studied and compared. N-cadherin, which was expressed by fusing myoblasts, was down-regulated on old myotubes while N-CAM was still present. Both molecules, as viewed by confocal microscopy, appeared to have coaccumulated at the areas of contact between fusing myoblasts. However, immunogold electron microscopy did not reveal significant colocalization of N-cadherin and N-CAM, and their segregation after antibody-induced patching suggested the absence of direct interactions between N-cadherin and N-CAM. The role of the Ca2+ dependent cell adhesion molecule N-cadherin in myogenesis was investigated. Myoblast fusion was inhibited (1) with a synthetic peptide containing the H-A-V sequence and (2) with a monoclonal anti-N-cadherin antibody, demonstrating that N-cadherin-mediated cell adhesion is required for myoblast fusion. Under the same conditions no effect of anti-N-CAM antibodies was observed. Taken together these observations suggest that N-cadherin, acting independently from N-CAM, is a major cell adhesion molecule involved in embryonic myoblast fusion in vitro .
A chimeric N-cadherin/β 1-integrin receptor which localizes to both cell-cell and cell-matrix adhesions
Authors: Geiger Benjamin , Salomon Daniela , Takeichi Masatoshi , Hynes Richard O.
Published: April 2021

To study the molecular mechanisms involved in formation of cell contacts, we have transfected cultured cells with a chimeric cDNA encoding the cytoplasmic and transmembrane domains of 1 integrin and the extracellular region of N-cadherin and determined the subcellular distribution of the chimeric molecule. We show that the chimeric receptor associates preferentially with cell-matrix focal contacts, suggesting that its distribution is directed by its 1 integrin segment, presumably via interactions of the cytoplasmic domain with cytoskeletal elements characteristic of focal contacts. Transfected cells which expressed relatively high levels of the cadherin/integrin chimera underwent an apparent epithelialization and contained the molecule both in cell-matrix and cell-cell contacts. Location in cell-cell contacts indicates competence of the cadherin extracellular domain to participate in formation of cell-cell junctions using a foreign cytoplasmic domain. Labeling of these cultures for talin, which is normally associated only with matrix adhesions, revealed specific labeling along the newly formed intercellular junctions. This suggests that the local association of talin with these sites is induced by the cytoplasmic tail of 1 integrin receptor presented by the chimeric protein. These results suggest that the formation of adherens-type junctions is driven by the cooperative interactions of the relevant adhesion molecules (cadherins and integrins) both with the respective extracellular ligands and with the cytoskeleton.
Nuclear proteins of the bovine esophageal epithelium: I. Monoclonal antibody W2 specifically reacts with condensed nuclei of differentiated superficial cells
Authors: Tang Tang K. , Hong Tse-Ming , Lin Chen-Yong , Lai Mei-Ling , Liu Catherine H. L. , Lo Hsiu-Jong , Wang Ming-Eng , Chen Lan Bo , Chen Wen-Tien , Ip Wallace , Lin Diane C. , Lin Jim J.-C. , Lin Shin , Sun Tung-Tien , Wang Eugenia , Wang John L. , Wu Reen , Wu Cheng-Wen , Chien Shu
Published: April 2021

Cells from three layers of the bovine esophageal epithelium, representing different stages of differentiation, were dissociated and separated by Percoll gradient centrifugation into fractions of small, medium and large sizes. A majority of the large cells possessed condensed nuclei, a characteristic feature of terminal differentiation of the superficial epithelium. The small cells resembled the proliferate cells of the basal layer. In vitro culture of the esophageal epithelial cells resulted in proliferation of the small cells, colony formation, and, in some cases, differentiation into cells with condensed nuclei. Nuclei, or nuclear subfractions derived from cells of the different layers, were used as immunogens for the generation of hybridomas secreting monoclonal antibodies that bound specifically to different regions of the esophageal tissue. One such antibody, designated W2, labeled the condensed nuclei from the superficial layer of stratified esophageal and corneal epithelia in situ, as well as the large cells from esophageal culture in vitro.

Thus, the expression of the W2 antigen may be associated with the process of nuclear condensation during epithelial differentiation. Immunoisolation of the target antigen of W2 from extracts of large cells of the bovine esophagus yielded a band of Mr ∼33,000 on nonreducing polyacrylamide gels. This band dissociated into two polypeptides, of Mr ∼22,000 and ∼11,000, upon treatment with dithiothreitol. Amino acid sequence analysis of the larger polypeptide showed extensive homology to a group of small calcium-binding proteins, including two helix-turn-helix motifs designated as the EF-hand, characteristic of the configuration of the metal-ion coordinating ligands of the calcium-binding site. Similarly, the sequence at the amino terminus of the polypeptide of ∼11,000 indicated that it was the light chain counterpart of the same calcium-binding protein complex.
Condensation of the chromatin at the membrane of an apoptotic nucleus is not associated with activation of an endonuclease
Authors: Oberhammer Franziska , Fritsch G. , Schmied M. , Pavelka M. , Printz D. , Purchio T. , Lassmann H. , Schulte-Hermann Rolf
Published: April 2021

A current hypothesis holds that chromatin fragmentation into oligonucleosomal patterns is an early event during apoptosis. In contrast, induction of apoptosis in cultured hepatocytes by TGF-β1 was not associated with DNA fragmentation into oligonucleosomes in hepatocyte monolayers and apoptotic fragments. For a more rigorous test of the hypothesis we performed a number of experiments. We compared nuclear changes resulting from TGF-β1 with those induced by Ca2+, a known activator of endonuclease. The morphology of apoptotic and Ca2+-treated nuclei was different as judged by DNA staining with Hoechst 33258. Likewise, electron microscopy of apoptotic nuclei showed characteristic condensation of the chromatin as well as dissolution of the nucleolar structure and nuclear fragmentation, changes not seen after Ca2+ treatment, after three hours of incubation. Analysis of DNA fluorescence of nuclei by FACS revealed that treatment with Ca2+ reduced the signal by 20%. In contrast, nuclei from TGF-β1-treated hepatocytes did not exhibit a reduced signal and after sorting by FACS, apoptotic nuclei remained in the 2N and 4N fractions. The absence of detectable DNA fragmentation in apoptotic nuclei was further verified by in situ nick translation, not only in hepatocytes but also in a mouse lymphoma cell line. From these findings we conclude that activation of an endonuclease is not an early event on the pathway to morphologically recognizable apoptosis.
Shapes of cells spreading on fibronectin: measurement of the stellation of BHK21 cells induced by raising cyclic AMP, and of its reversal by serum and lysophosphatidic acid
Authors: Edwards J. G. , Campbell G. , Carr M. , Edwards C. C.
Published: April 2021

In common with many other animal cells in culture, BHK21, CHO and NIH-3T3 cells adopt bizarre stellate or arborized shapes when exposed, in the absence of serum, to agents which increase cytoplasmic cyclic AMP (cAMP). Dibutyryl cAMP, 3-isobutyl-1-methylxanthine, 5′-deoxy-5′-methylthioadenosine, cholera toxin and the invasive adenylate cyclase from Bordetella pertussis all induce similar shapes. Time lapse video recording of BHK21 cells spreading on fibronectin shows that stellate shapes are generated by outgrowth of neurite-like processes led by small fans of ruffling membrane. These structures stain strongly for F actin, and their out-growth is completely inhibited by cytochalasin D. Thus if stellation is caused by microfilament depletion, this must be selective for subsets of microfilaments.

We have quantified the shape changes of BHK21 cells using the parameter dispersion. They are prevented by low concentrations (1% by volume and below) of bovine sera. The inhibitory component of foetal bovine serum acts humorally, behaves as a macromolecule and is itself inhibited by suramin, but platelet-derived growth factor, insulin, vasopressin and bradykinin are inactive. The inhibitory activity of serum may be due to phos-pholipids, since it can be replaced by lysophosphatidic acid in the presence of serum albumin.
EMAP, an echinoderm microtubule-associated protein found in microtubule-ribosome complexes
Authors: Suprenant Kathy A. , Dean Kathleen , McKee Julie , Hake Shelly
Published: April 2021

The major non-tubulin polypeptide found associated with microtubules purified from unfertilized sea urchin eggs by cycles of pH-dependent assembly has a Mr of 77,000. The 77,000 Mr polypeptide is heat- and acidlabile, and is antigenically distinct from the mammalian brain MAPs, MAP-2 and tau. Affinity-purified antiserum against the 77,000 Mr polypeptide was used to survey a variety of cells and tissues for the presence of antigenically related polypeptides. A cross-reacting polypeptide, ranging in Mr from 72,000 to 80,000, was found in microtubule preparations from a wide variety of echinoderms, including sea urchins, starfish and sand dollars. Indirect immunofluorescence showed that the polypeptide was found in interphase as well as mitotic microtubule arrays. No cross-reacting material was detected in microtubules isolated from marine molluscs, mammalian brain or mouse B16 cultured cells. Because the 77,000 Mr MAP is abundant in echinoderms, we have called it EMAP for echinoderm microtubule-associated protein. Although the precise function of the EMAP is not known, our data suggest that the EMAP is involved in the attachment of ribosomes to microtubules. Large numbers of ribosomes are attached to the walls of EMAP-containing microtubules, but not EMAP-deficient microtubules. Removal of the EMAP from the microtubule by salt-extraction results in the release of ribosomes from the microtubule, indicating that the EMAP may form part or all of the long tapered stalk that connects these two organelles.
Mycelial and syncytial growth in Schizosaccharomyces pombe induced by novel septation mutations
Authors: Sipiczki M. , Grallert B. , Miklos I.
Published: April 2021

Mutation in the gene sep1+ of the unicellular fission yeast Schizosaccharomyces pombe impairs cell separation after cytokinesis and confers a branching mycelial mor­phology. The mutant is not defective in cell wall β-glu- canase activity but shows increased sensitivity to Ca2+ and Mg2+, and increased resistance to the microtubule inhibitor benomyl. The mycelial growth of sep1-1 pro­vides a convenient method for the examination of the polar growth pattern and for pedigree analysis as demonstrated by the segregation of mating types in the homothallic microhyphae. sepl is closely linked to adel (0.94 cM) on the right arm of chromosome II. The ts mutation spl1-1 confers a bent cell shape and causes aberrant septum formation at the restrictive tempera­ture. sep1+ and spl1+ perform closely related functions as their mutant alleles interact with each other and with another septation mutant cdc4-8. These functions may overlap with certain cytoskeletal processes and with the determination of cell polarity because the triple mutant forms huge multinucleate syncytia with promiscuous branching and rare septum formation.
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

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

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

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

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

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

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 >60% of the cell population after 60 min and >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 >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

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

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

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

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

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

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

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.