bluuebunny/crossref_metadata_2025_split huggingface dataset:
Distribution of hyaluronan in the epiphysial growth plate: turnover by CD44-expressing osteoprogenitor cells
Authors: Pavasant Prasit , Shizari Tony M. , Underhill Charles B. 
Published: April 2021

ABSTRACT
In the present study, we have examined the distribution of both hyaluronan and its receptor, CD44, during the process of endochondral ossification in the mouse tibia. Histochemical staining revealed that a large amount of hyaluronan was present in the lacunae located in the zone of hypertrophy, but it was greatly reduced or absent from the zone of erosion. In addition, hyaluronan was present in the cytoplasm of osteoprogenitor cells located in the zone of erosion. These cells also expressed CD44 on their surfaces, as revealed by double-label immunohistochem-istry. These results suggested that the osteoprogenitor cells may use CD44 to bind and internalize hyaluronan, and subsequently degrade it with lysosomal enzymes. To test this possibility, we examined the human cell line, MG-63, which closely resembles osteoprogenitor cells. These cells produced several different forms of CD44, as determined by western blotting (85, 116 and 150 kDa). In addition, the binding of isotopically labeled hyaluronan to detergent extracts of these cells was blocked by a monoclonal antibody to CD44. Similarly, the degradation of hyaluronan by these cultured cells was also inhibited by a mono-clonal antibody to CD44. To determine if these cells could remove hyaluronan from the growth plate, the cells were cultured directly on top of thin sections of the epiphysial region of long bone. After 16 hours, the sections were stained for hyaluronan. The MG-63 cells removed signifi-cant amounts of hyaluronan present in the zone of hyper-trophy, and this effect was blocked by an excess of soluble hyaluronan and by a monoclonal antibody to CD44. Taken together, these results suggest that, under physiological conditions, osteoprogenitor cells in the zone of erosion can use a CD44-dependent mechanism to remove hyaluronan.
Programmed cell death in Dictyostelium
Authors: Cornillon Sophie , Foa Colette , Davoust Jean , Buonavista Nathalie , Gross Julian D. , Golstein Pierre 
Published: April 2021

ABSTRACT
Programmed cell death (PCD) of Dictyostelium discoideum cells was triggered precisely and studied quantitatively in an in vitro system involving differentiation without morphogenesis. In temporal succession after the triggering of differentiation, PCD included first an irreversible step leading to the inability to regrow at 8 hours. At 12 hours, massive vacuolisation was best evidenced by confocal microscopy, and prominent cytoplasmic condensation and focal chromatin condensation could be observed by electron microscopy. Membrane permeabilization occurred only very late (at 40-60 hours) as judged by propidium iodide staining. No early DNA fragmentation could be detected by standard or pulsed field gel electrophoresis. These traits exhibit some similarity to those of previously described non-apoptotic and apoptotic PCD, suggesting the hypothesis of a single core molecular mechanism of PCD emerging in evolution before the pos-tulated multiple emergences of multicellularity. A single core mechanism would underly phenotypic variations of PCD resulting in various cells from differences in enzymatic equipment and mechanical constraints. A pre-diction is that some of the molecules involved in the core PCD mechanism of even phylogenetically very distant organisms, e.g. Dictyostelium and vertebrates, should be related.
Synaptonemal complex proteins: occurrence, epitope mapping and chromosome disjunction
Authors: Dobson Melanie J. , Pearlman Ronald E. , Karaiskakis Angelo , Spyropoulos Barbara , Moens Peter B. 
Published: April 2021

ABSTRACT
We have used polyclonal antibodies against fusion proteins produced from cDNA fragments of a meiotic chromosome core protein, Cor1, and a protein present only in the synapsed portions of the cores, Syn1, to detect the occurrence and the locations of these proteins in rodent meiotic prophase chromosomes. The 234 amino acid Cor1 protein is present in early unpaired cores, in the lateral domains of the synaptonemal complex and in the chromosome cores when they separate at diplotene. A novel observation showed the presence of Cor1 axial to the metaphase I chromosomes and substantial amounts of Cor1 in association with pairs of sister centromeres. The centromere-associated Cor1 protein becomes dissociated from the centromeres at anaphase II and it is not found in mitotic metaphase centromeres. The extended presence of Cor1 suggests that it may have a role in chromosome disjunction by fastening chiasmata at metaphase I and by joining sister kinetochores, which ensures co-segregation at anaphase I. Two-colour immunofluorescence of Cor1 and Syn1 demonstrates that synapsis between homologous cores is initiated at few sites but advances rapidly relative to the establishment of new initiation sites. If the rapid advance of synapsis deters additional initiation sites between pairs of homologues, it may provide a mechanism for positive recombi- nation interference. Immunogold epitope mapping of anti- bodies to four Syn1 fusion proteins places the amino terminus of Syn1 towards the centre of the synaptonemal complex while the carboxyl terminus extends well into the lateral domain of the synaptonemal complex. The Syn1 fusion proteins have a non-specific DNA binding capacity. Immunogold labelling of Cor1 antigens indicates that the lateral domain of the synaptonemal complex is about twice as wide as the apparent width of lateral elements when stained with electron-dense metal ions. Electron microscopy of shadow-cast surface-spread SCs confirms the greater width of the lateral domain. The implication of these dimensions is that the proteins that comprise the synaptic domain overlap with the protein constituents of the lateral domains of the synaptonemal complex more than was apparent from earlier observations. This arrangement suggests that direct interactions might be expected between some of the synaptonemal complex proteins.
The role of duplication of tumour-derived chromosome 15 carrying the rearranged pvt-1 gene in the transformed phenotype of YACUT T-cell lymphoma × G4 T-cell line somatic cell hybrids in dictating the terminal differentiation program of the parental G4 cell
Authors: Kubota Koichi , Nakazato Kazumi 
Published: April 2021

ABSTRACT
Fusion of the YACUT T-cell lymphoma with the Mls-1a-antigen-specific non-tumorigenic T-cell line G4 was previously reported to produce growth-arrested hybrids that could be induced to proliferate in the presence of Mls-1a antigen. The proliferation-suppressed hybrid lines exhibited phenotypic changes as follows: the usually high levels in YACUT of J11d antigen, IL-2 receptor, and c-myb expression, which are markers of immature T cells, were all down-regulated; the G4 T-cell function, i.e., contact helper activity for B-cell proliferation in T/B cell collabo-ration, was retained. Furthermore, fusion of the YACUT lymphoma with a killer T-cell line produced growth-arrested and tetraploid somatic cell hybrids having killer activity. Thus, in addition to the transformed phenotype (autonomous proliferation in vitro), the antigen-specific non-tumorigenic T-cell line genomes introduced into the YACUT lymphoma suppressed the immature phenotypes of YACUT and imposed their own programming of terminally differentiated traits on the hybrids.
Prolonged growth of the proliferation-suppressed hybrid lines by repeated antigenic stimulation was previously reported to result in the appearance of transformed hybrids, which was accompanied by both a reversion of c-myc expression to the levels of YACUT and an increase in the number of chromosome 15. The present study revealed that the amplification of chromosome 15 resulted from the duplication of the tumour-derived chromosome 15 carrying the rearranged pvt-1 gene. However, the differentiated phenotypes of the hybrids remained mostly unchanged upon cell transformation. These results indicate that the duplication of the tumour-derived chromosome 15 plays an important role in the appearance of the trans-formed phenotype without abrogating the imposition of the terminally differentiated traits on the YACUT × G4 hybrids.
Differential effects of compartment deacidification on the targeting of membrane and soluble proteins to the vacuole in yeast
Authors: Morano Kevin A. , Klionsky Daniel J. 
Published: April 2021

ABSTRACT
Lysosomal/vacuolar protein targeting is dependent on compartment acidification. In yeast, sorting of soluble vacuolar proteins such as carboxypeptidase Y is sensitive to acute changes in vacuolar pH. In contrast, the vacuolar membrane protein alkaline phosphatase is missorted only under conditions of chronic deacidification. We have undertaken a temporal analysis to define further the relationship between compartment acidification and sorting of soluble and membrane vacuolar proteins. Depletion of either the Vma3p or Vma4p subunits of the yeast vacuolar ATPase over time resulted in loss of vacuolar ATPase activity and vacuolar acidification. A kinetic delay in processing of carboxypeptidase Y occurred concomitant with these physiological changes while transport of alkaline phosphatase remained unaffected. Carboxypeptidase S, another vacuolar hydrolase that transits through the secretory pathway as an integral membrane protein, displayed a pH sensitivity similar to that of soluble vacuolar proteins. These results indicate that compartment acidification is tightly coupled to efficient targeting of proteins to the vacuole and that there may be multiple distinct mechanisms for targeting of vacuolar membrane proteins.
Recruitment of antigenic gamma-tubulin during mitosis in animal cells: presence of gamma-tubulin in the mitotic spindle
Authors: Lajoie-Mazenc Isabelle , Tollon Yvette , Detraves Claire , Julian Monique , Moisand André , Gueth-Hallonet Catherine , Debec Alain , Salles-Passador Isabelle , Puget Alain , Mazarguil Honoré , Raynaud-Messina Brigitte , Wright Michel 
Published: April 2021

ABSTRACT
It has been claimed repeatedly that γ-tubulin is exclusively localized at the spindle poles in mitotic animal cells, where it plays a role in microtubule nucleation. In addition to this localization, we have observed a γ-tubulin-specific staining of the mitotic spindle in several animal cells (human, kangaroo rat, mouse, Chinese hamster, Xenopus and Drosophila) using five polyclonal antibodies raised against unique γ-tubulin sequences and four different fixation protocols. In HeLa and PtK2 cells, γ-tubulin was detected in the mitotic spindle from late prometaphase to telophase. In contrast, in other cell types, it was detected in metaphase only. In all cases we failed to detect γ-tubulin in the short aster microtubules at the spindle poles. Electron microscopic observation revealed that at least part of the γ-tubulin localized on the surface of spindle microtubules with a preferential distribution along kinetochore microtubules. In HeLa cells, the amount of antigenic γ-tubulin was fairly constant in the spindle poles during mitosis from prometaphase to telophase. In contrast, γ-tubulin appeared in the mitotic spindles in prometaphase. The amount of γ-tubulin decreased in telophase, where it relocalized in the interzone. In metaphase cells about 15-25% of the total fluorescence was localized at the spindle poles, while 75-85% of the fluorescence was distributed over the rest of the spindle. These results suggest that the localization and timing of γ-tubulin during the cell cycle is highly regulated and that its physiological role could be more complex and diverse than initially assumed.
TRiC-P5, a novel TCP1-related protein, is localized in the cytoplasm and in the nuclear matrix
Authors: Joly Erik C. , Tremblay Esther , Tanguay Robert M. , Wu Yang , Bibor-Hardy Viviane 
Published: April 2021

ABSTRACT
We have recently reported the cloning of a novel protein, TRiC-P5, with significant homology with protein 1 of the t-complex (TCP1). In the present study, the cellular localization of TRiC-P5 in Raji cells has been determined using an antiserum raised against a 18.5 kDa fusion protein. Results from cell fractionation and immunoblot studies indicate that TRiC-P5 is mainly localized in the cytoplasm. In addition, a significant part of TRiC-P5 is also found in the nucleus where it is attached to the nuclear matrix, a complex filament network involved in essential cellular functions such as DNA replication, and RNA transcription and maturation. Immunofluorescence experiments using the anti-TRiC-P5 antibodies confirm these results. We also provide evidence that, in the cytoplasm, TRiC-P5 is part of a large protein complex, most probably the TCP1-ring complex (TRiC), a hetero-oligomeric ring complex that plays a role of molecular chaperone in the folding of actin and tubulin.
Topoisomerase II expression and VM-26 induction of DNA breaks during spermatogenesis in Xenopus laevis
Authors: Morse-Gaudio Michele , Risley Michael S. 
Published: April 2021

ABSTRACT
The relative content of topoisomerase II (topo II) and the induction of topo-II-mediated DNA damage and cellular abnormalities have been characterized in developing sper-matogenic cells of Xenopus laevis to gain an insight into the roles of topo II during spermatogenesis. Decatenation assays identified topo II activity in nuclear extracts from spermatocytes and pre-elongate spermatids, but not in extracts from elongate spermatids or sperm. Extracts from early-mid spermatids contained 14% (per cell) of the decatenation activity found in spermatocyte extracts. Immunoblots of SDS extracts from whole cells and nuclei from both spermatocytes and pre-elongate spermatids, but not elongate spermatids or sperm, resolved a 180 kDa polypeptide that reacts with polyclonal antisera to Xenopus oocyte topo II, an antipeptide antibody (FHD29) to human topo IIα and β, and an antipeptide antibody to human topo IIα, suggesting homology between Xenopus spermatogenic cell topo II and mammalian topo IIα. Immunofluorescence microscopy of topo II in testis cryosections revealed the presence of topo II in nuclei of all spermatogenic stages, but not in sperm. The relative levels of topo II estimated from fluorescence intensity were highest in spermatogonia and spermatocytes, then early-mid spermatids, followed by elongate spermatids and somatic cells. Incubation of isolated spermatogenic cells with teniposide (VM-26), a topo II-targetted drug, resulted in a dose-dependent induction of DNA breaks in all spermatocytes and spermatid stages to nuclear elongation stages, as analyzed by alkaline single cell gel electrophoresis. Addition of 0.5-50 μM VM-26 to spermatogenic cell cultures for 27 hours resulted in stage-dependent abnormalities. Mid-late spermatid stages were relatively resistant to VM-26-induced damage. In contrast, meiotic division stages were arrested and spermatogonia B were killed by VM-26, and VM-26 induced abnormal chromosome condensation in pachytene spermatocytes. The results of these studies show that cellular levels of topo II are stage-dependent during spermatogenesis, that most spermatogenic stages are sensitive to topo II-mediated DNA damage, and that spermatogonia B, meiotic divisions and pachytene spermatocytes are particularly sensitive to induction of morphological abnormalities and cell death during acute exposure to topo II-targetted drugs.
cAMP does not regulate [Ca2+]i in human tracheal epithelial cells in primary culture
Authors: Davis Pamela B. , Perez Catherine L. Silski and Aura 
Published: April 2021

ABSTRACT
Human tracheal epithelial cells in primary culture respond to different receptor agonists with different peak intracel- lular calcium concentrations. From resting concentration 138±13 nM, bradykinin (0.1 μM) produces an increase to a maximum of 835±195 nM, histamine (10 μM) to 352±51 nM, and ATP (5-500 μM) to more than 1500 nM. Nine of 14 cultures also responded to isoproterenol (10 μM), though with a smaller increase, to 210±29 nM. A response was observed with isoproterenol, and epinephrine, but not norepinephrine, phenylephrine or methoxamine, was inhibited by propranolol but not phentolamine, and so this appeared to be a β-adrenergic response. However, no response could be detected to adenosine, prostaglandin E2 or forskolin, agents that activate adenylate cyclase, or to permeant analogs of cAMP (CPT-cAMP or db-cAMP). The intracellular calcium response to isoproterenol did not follow either the time-course or the desensitization pattern of the cAMP response. Thus, this response to isoproterenol is not mediated by cAMP. No relation was demonstrated between cAMP production by other agonists and the response of intracellular calcium. Pretreatment with agents that increase cAMP did not affect the calcium responses to ATP or bradykinin. Thus, cAMP does not regulate intra- cellular calcium concentration in human tracheal epithelial cells. The variation in peak intracellular calcium responses to various agonists may be explained by the presence of multiple second messengers (other than cAMP), multiple intracellular pools of calcium, or cell heterogeneity. The agonists tested had the same relative potency in cells from patients with cystic fibrosis as in non-cystic fibrosis cells.
The network organization and the phosphorylation of cytokeratins are concomitantly modified by forskolin in the enterocyte-like differentiated Caco-2 cell line
Authors: Baricault Laurent , Néchaud Béatrice de , Sapin Catherine , Codogno Patrice , Denoulet Philippe , Trugnan Germain 
Published: April 2021

ABSTRACT
Confluent Caco-2 cells, originating from a human colon carcinoma, display morphological and functional charac-teristics of differentiated enterocytes such as the presence of a polarized monolayer covered by an apical brush border that express several hydrolases. The adaptation of these cells to grow in the continuous presence of forskolin, a drug known to stimulate adenylyl cyclase permanently, has been previously shown to result in a decreased apical expression of hydrolases and in morphological alterations including the disappearance of intercellular spaces and shortening of microvilli. In the present work we have analyzed the possibility that cytoskeletal proteins may be the target of forskolin in living Caco-2 cells. We show that forskolin initiates dramatic changes in the spatial organiz-ation of the cytokeratin network that correlate with an increased phosphorylation of cytokeratin molecules, whereas microtubules, microfilaments and vimentin remain mainly unaffected. Indirect immunofluorescence studies show that the cytokeratin network is redistributed from the cell periphery to the cytoplasm. Biochemical experiments indicate that forskolin doesn’t interfere with the cytokeratin profile, since the three cytokeratins normally found in intestine (CK 8, CK 18, CK 19) are similarly expressed in both control and forskolin-Caco-2 cells. Analysis of 32P-labeled cytokeratin extracted from the two cell populations demonstrates that forskolin quantita-tively increases the phosphorylation of type I cytokeratin (CK 18 and CK 19), whereas the phosphorylation of type II cytokeratin (CK 8) is altered both quantitatively and qualitatively with the emergence of a new phosphorylation site. These results provide a new cell system in which it is possible to control the subcelluar distribution of cytoker-atin by changing their phosphorylation status and therefore to study their potential cellular functions.
Ectoenzyme regulation by phenotypically distinct fibroblast sub-populations isolated from the human mammary gland
Authors: Atherton Amanda J. , O’Hare Michael J. , Buluwela Lakjaya , Titley Jenny , Monaghan Paul , Paterson Hugh F. , Warburton Michael J. , Gusterson Barry A. 
Published: April 2021

ABSTRACT
Inter- and intralobular mammary fibroblasts have been separated from normal human breast tissue and cultured to study the differential expression of ectoenzymes present within the stroma of the normal gland and associated with breast cancers. Specific ectoenzymes were identified by indirect immunofluorescence and quantified by flow cytometry and semi-quantitative PCR. A consistent difference was noted between the two fibroblast sub-populations at early passage in respect of dipeptidyl peptidase IV (DPP IV) and aminopeptidase N (APN) expression. Early passage intralobular fibroblasts were positive for APN but negative for DPP IV, as seen in the intact tissue. However, with continued sub-culture they gradually began to express DPP IV, until at later passages they became indistinguishable from the interlobular fibroblasts, which were APN and DPP IV-positive at all stages in culture, as they are in intact tissue. Neutral endopeptidase (NEP/CALLA/CD10) is not expressed by normal adult breast fibroblasts but is found in the stroma associated with over 60% of breast cancers.
It was up-regulated in vitro on both inter- and intralobular fibroblasts, with final levels that were significantly (&lt;14 times) higher on the former in all pairs of preparations from individual donors analysed. This difference persisted with continued passage, and levels of the ectoenzyme and its messenger RNA were further up-regulated by hydro-cortisone in both populations. These results demonstrate that phenotypically distinct cultures of human mammary fibroblast sub-populations can be used to study the regulation of these stromal ectoenzymes. Such cultures also provide a means of investigating the mechanisms and functional significance of changes occurring as the result of neoplasia, as well as enabling the individual roles of inter- and intralobular fibroblasts in epithelial/mesenchymal interactions to be evaluated.
Dissociation of minor satellite from the centromere in mouse
Authors: Vig Baldev K. , Latour Derek , Frankovich Jennifer 
Published: April 2021

ABSTRACT
The minor satellite DNA of mouse is believed to constitute the centromere. We report that centromeres of some chromosomes in the Cl1D cells of mouse are not associated with this DNA even though the latter is present on these chromosomes. The satellite DNA was detected distally from the centromere and could not be mistaken as a component of the centromere. We also report that the site of the primary constriction may not always coincide with the site of the anti-kinetochore antibody reaction. Whereas the regions containing the major satellite decondense upon treatment with bisbenzimidazole (Hoechst 33258), the sites carrying minor satellite resist decondensing.
Overexpression of a truncated cyclin B gene arrests Dictyostelium cell division during mitosis
Authors: Luo Qian , Michaelis Christine , Weeks Gerald 
Published: April 2021

ABSTRACT
A cyclin gene has been isolated from Dictyostelium discoideum and the available evidence indicates that the gene encodes a B type cyclin. The cyclin box region of the protein encoded by the gene, clbl, has the highest degree of sequence identity with the B-type cyclins of other species. Levels of cyclin B mRNA and protein oscillate during the cell cycle with maximum accumulation of mRNA occurring prior to cell division and maximum levels of protein occurring during cell division. Overexpression of a N-ter- minally truncated cyclin B protein lacking the destruction box inhibits cell growth by arresting cell division during mitosis. The gene is present as a single copy in the Dictyostelium genome and there is no evidence for any other highly related cyclin B genes.