bluuebunny/crossref_metadata_2025_split huggingface dataset:
Decrease in nuclear phospholipids associated with DNA replication
Authors: Maraldi Nadir M. , Santi Spartaco , Zini Nicoletta , Ognibene Andrea , Rizzoli Riccardo , Mazzotti Giovanni , Primio Roberto Di , Bareggi Renato , Bertagnolo Valeria , Pagliarini Carla , Capitani Silvano 
Published: April 2021

ABSTRACT
Lipid metabolism in nuclei is very active and appears involved in the transduction of signals to the genome in response to agonists acting at the plasma membrane level. However, the precise topology of nuclear lipid metabolism and the relationship between nuclear lipids and crucial events of the cell function, such as DNA replication, have not been fully elucidated. By using a recently developed cytochemical method for detecting phospholipids inside the nucleus of intact cells at the electron microscope level, we have analyzed the changes in intranuclear phospholipids in DNA-replicating versus resting cells, which are both present in the same sample of regenerating liver after partial hepatectomy.
The pattern of DNA synthesis in replicating cells has been monitored by electron microscope immunocytochemistry after bromodeoxyuridine (BrdU) labeling. The data obtained, which allow a fine localization and a quantitative analysis of both DNA synthesis and phospholipid distribution, indicate a significant reduction in the phospholipids detectable inside the nucleus in all steps of the S phase. This could depend on an increased nuclear phospholipid hydrolysis, whose products should in turn activate some of the enzymes involved in the control of DNA replication.
Inhibition of protein kinases by 6-dimethylaminopurine accelerates the transition to interphase in activated mouse oocytes
Authors: Szöllösi Maria S. , Kubiak Jacek Z. , Debey Pascale , Pennart Henri de , Szöllösi Daniel , Maro Bernard 
Published: April 2021

ABSTRACT
Mouse oocyte activation is followed by a peculiar period during which the interphase network of microtubules does not form and the chromosomes remain condensed despite the inactivation of MPF. To evaluate the role of protein phosphorylation during this period, we studied the effects of the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP) on fertilization and/or parthenogenetic activation of metaphase II-arrested mouse oocytes. 6-DMAP by itself does not induce the inactivation of histone H1 kinase in metaphase II-arrested oocytes, and does not influence the dynamics of histone H1 kinase inactivation during oocyte activation. However, 6-DMAP inhibits protein phosphorylation after oocyte activation. In addition, the phosphorylated form of some proteins disappear earlier in oocytes activated in the presence of 6-DMAP than in the activated control oocytes. This is correlated with the acceleration of some post-fertilization morphological events, such as sperm chromatin decondensation and its transient recondensation, formation of the interphase network of microtubules and pronuclear formation. In addition, numerous abnormalities could be observed: (1) the spindle rotation and polar body extrusion are inhibited; (2) the exchange of protamines into histones seems to be impaired, as judged by the morphology of DNA fibrils by electron microscopy; (3) the formation of a new nuclear envelope around the sperm chromatin proceeds prematurely, while recondensation is not yet completed. These observations suggest that the 6-DMAP-sensitive kinase(s) is (are) involved in the control of post-fertilization events such as the formation of the interphase network of microtubules, the remodelling of sperm chromatin and pronucleus formation.
Inactivation of cdc2 kinase during mitosis requires regulated and constitutive proteins in a cell-free system
Authors: Suprynowicz Frank A. 
Published: April 2021

ABSTRACT
Inactivation of the cyclin-p34cdc2 protein kinase complex is a major requirement for anaphase onset and exit from mitosis. To facilitate identification of specific molecules that regulate this event in mammalian cells, I have developed a cell-free assay in which cdc2 kinase associated with a chromosomal fraction from metaphase tissue culture cells is inactivated by a cell-cycle-regulated cytosolic system. In vitro kinase inactivation requires ATP, Mg2 +and the dephosphorylation of one or more sites in the chromosomal fraction by protein phosphatase 1 and/or 2A. Cyclin B is destroyed during inactivation, while the level of p34cdc2 remains constant. Ammonium sulfate fractionation resolves the cytosolic inactivating system into at least two distinct protein components that are both required for inactivation and are differentially regulated during mitosis.
Cytoskeleton interactions in the ascus development and sporulation of Sordaria macrospora
Authors: Thompson-Coffe Catherine , Zickler Denise 
Published: April 2021

ABSTRACT
The organization of actin during meiosis and sporulation in the ascus of the filamentous ascomycete Sordaria macrospora was determined by immunofluorescence without removal of the cell wall. Actin is present as a dense cortical network of microfilaments (MF) and plaques, a perinuclear shell of actin in prophase I of meiosis, and a complex array of MF involved in alignment of prespore nuclei and closure of spore cell membranes. The relationship of actin to the previously examined microtubule system of the ascus was determined by double-label immunofluorescence. The cytoskeletal inhibitors nocodazole, cytochalasin D and 2,3-butanedione monoxime were used to examine the roles of actin and myosin in ascus development. Microfilament and microtubule arrays are interdependant; disruption of one network results in abnormalities in the other. Both microfilaments and actin-myosin interaction are required for separation and migration of duplicated spindle pole bodies, septation and sporulation.
Purification of proteasomes from salmonid fish sperm and their localization along sperm flagella
Authors: Inaba Kazuo , Akazome Yasuhisa , Morisawa Masaaki 
Published: April 2021

ABSTRACT
We have purified two chymotrypsin-like proteases from chum salmon sperm which have no apparent acrosome structure. Both of them were high molecular mass proteases (650 kDa and 950 kDa by gel filtration) and showed not only chymotrypsin-like activity but also trypsin-like activity. The 650 kDa protease was composed of at least eight or nine kinds of polypeptide with molecular masses ranging from 20 kDa to 30 kDa and was highly activated by low concentrations of SDS. Electron microscopy revealed that the 650 kDa protease was a ring-shaped particle. The 950 kDa protease was shown to contain at least one component that cross-reacts with an antibody against the 650 kDa protease. Finally, we revealed that the 650 kDa protease is located along the sperm flagella, by using immunofluorescence microscopy. The subunit composition, SDS-activation and molecular shape of 650 kDa salmonid protease were quite similar to those of the eukaryotic multicatalytic proteinase (proteasome), which is well known to participate in ATP-dependent degradation of ubiquitinated proteins; and, furthermore, the motility of demembranated sperm of salmonid fish is inhibited by chymotrypsin inhibitors in an ATP-dependent manner. Thus, the protease located in salmonid fish sperm flagella is a proteasome and is a strong candidate for the factor which regulates flagellar motility in an ATP-dependent manner.
Nanomelic chondrocytes synthesize, but fail to translocate, a truncated aggrecan precursor
Authors: Vertel Barbara M. , Walters Linda M. , Grier Bonnie , Maine Nancy , Goetinck Paul F. 
Published: April 2021

ABSTRACT
Cartilage extracellular matrix (ECM) is composed primarily of type II collagen and large, link stabilized aggregates of hyaluronic acid and chondroitin sulfate proteoglycan (aggrecan). Maturation and function of these complex macromolecules are dependent upon sequential processing events which occur during their movements through specific subcellular compartments in the constituitive secretory pathway. Failure to complete these events successfully results in assembly of a defective ECM and may produce skeletal abnormalities. Nanomelia is a lethal genetic mutation of chickens characterized by shortened and malformed limbs. Previous biochemical studies have shown that cultured nanomelic chondrocytes synthesize a truncated aggrecan core protein precursor that disappears with time; however, the protein does not appear to be processed by the Golgi or secreted. The present study investigates the intracellular trafficking of the defective aggrecan precursor using immunofluorescence, immunoelectron microscopy and several inhibitors. Results indicate that nanomelic chondrocytes assemble an ECM that contains type II collagen, but lacks aggrecan. Instead, aggrecan precursor was localized intracellularly, within small cytoplasmic structures corresponding to extensions of the endoplasmic reticulum (ER). At no time were precursor molecules observed in the Golgi. In contrast, normal and nanomelic chondrocytes exhibited no difference in the intracellular or extracellular distribution of type II procollagen. Therefore, retention of the aggrecan precursor appears to be selective. Incubation of chondrocytes at 15°C resulted in the retention and accumulation of product in the ER. After a return to 37°C, translocation of the product to the Golgi was observed for normal, but not for nanomelic, chondrocytes, although the precursors disappeared with time. Ammonium chloride, an inhibitor of lysosomal function, had no effect on protein loss, suggesting that the precursor was removed by a non-lysosomal mechanism, possibly by ER-associated degradation. Based on these studies, we suggest that nanomelic chondrocytes are a useful model for examining cellular trafficking and sorting events and the processes by which abnormal products are targeted for retention or degradation. Further investigations should provide insight into the mechanisms underlying chondrodystrophies and other related diseases.
Expression of osteopontin mRNA by osteoclasts and osteoblasts in modelling adult human bone
Authors: Merry Karen , Dodds Robert , Littlewood Amanda , Gowen Maxine 
Published: April 2021

ABSTRACT
Over recent years several non-collagenous matrix proteins of bone have been isolated and characterized. One of these proteins, osteopontin, has been shown to be synthesized by osteoblasts and deposited in the bone matrix where it is thought to bind to hydroxyapatite. However much of the functional evidence is circumstantial, and the precise function of osteopontin has not been fully elucidated.
We have used in situ hybridization techniques to investigate the expression of osteopontin mRNA in a variety of human bone tissues. Cryostat sections of human osteophyte and osteoclastoma tissue were hybridized with an antisense RNA probe for osteopontin. Sense transcripts were used as a negative control to assess non-specific binding.
There was a very distinct pattern of osteopontin mRNA expression in these tissues. Plump osteoblasts adjacent to the osteoid matrix expressed high levels of osteopontin mRNA, whilst flattened osteoblasts demonstrated weaker expression. The most striking feature of osteopontin mRNA expression was the high levels detected in osteoclasts. Osteoclasts in resorption lacunae and those distant from resorption sites both expressed osteopontin mRNA, suggesting that attachment was not a prerequisite for osteopontin expression. A population of mononuclear cells in resorption lacunae was also observed to express high levels of osteopontin mRNA. The whole population of osteoclasts in the osteoclastoma tissue expressed high levels of osteopontin mRNA, indicating that expression is not restricted to osteoclasts involved in bone resorption.
This study confirms that human osteoblasts are capable of synthesizing osteopontin. However it highlights the fact that osteoclasts may not be dependent on osteopontin present in the matrix for attachment, as they are capable of synthesizing their own osteopontin to facilitate anchorage to the bone matrix prior to resorption.
Diverse mechanisms for cell attachment to platelet thrombospondin
Authors: Adams Josephine C. , Lawler Jack 
Published: April 2021

ABSTRACT
Thrombospondin-1 is a component of the extracellular matrix which is thought to play important roles in cell migration and proliferation, during embryogenesis and wound repair. To understand the basis for these activities, we are mapping the regions of the molecule with cell adhesive activity. Here, we use antagonists of specific cell binding sites, adhesion-perturbing throm-bospondin monoclonal antibodies and proteolytic fragments of platelet thrombospondin, to investigate the adhesive mechanisms used by G361 melanoma cells, human intestinal smooth muscle cells (HISM), epidermal keratinocytes and MG-63 osteosarcoma cells. When attached to the same preparations of platelet thrombospondin, HISM and MG-63 cells underwent spreading, whereas G361 cells and keratinocytes did not. Attachment of all four cell types involved the carboxy-terminal domain. The type 1 repeats and the amino-terminal heparin binding domain were important for stable attachment of G361, HISM and MG-63 cells, but were not involved in keratinocyte attachment. GRGDSP peptide caused near complete inhibition of HISM and MG-63 cell attachment, partially inhibited G361 attachment, but did not inhibit keratinocyte attachment. Attachment of HISM and MG-63 cells involved the αvα3 integrin. The integrity of the thrombospondin molecule was important for its adhesivity towards G361, HISM, and MG-63 cells, whereas keratinocytes attached to the 140 kDa tryptic fragment as effectively as they did to the intact molecule. These results show that cell attachment to platelet thrombospondin typically involves multiple binding interactions, but the exact profile of interactions is cell type specific. Usage of particular cell-binding sites does not predict whether cells will undergo spreading or not. These data may, in part, explain some of the current controversies surrounding the mechanisms of cell attachment to thrombospondin.
Abbreviations: TSP-1, thrombospondin-1; TSP-2, thrombospondin-2; TBS, Tris-buffered saline; DMEM, Dulbecco’s modified Eagle’s medium
The characteristic which makes the cell-coded HSV-inducible U90 distinctive in transformed cells is its greatly increased half-life
Authors: Grassie M. , McNab D. , Macnab J. C. M. 
Published: April 2021

ABSTRACT
A set of polypeptides detected in transformed cells of Mr values 90,000 (a doublet) 40,000 and 32,000 that are recognised by immunoprecipitation of L-[35S]methionine-labelled tumour cell lysates, with sera from tumour-bearing rats and with antisera raised against herpes simplex virus type 2 (HSV-2)-infected cells, were previously reported (Macnab et al., 1985, 1992; Hewitt et al., 1991). These proteins are cell-coded and cannot be pre-cipitated from similarly radiolabelled control cells. Two of these polypeptides are significantly induced by HSV-2 infection (Hewitt et al., 1991; Macnab et al., 1992). This paper further characterises one of these polypeptides, U90, and addresses which properties distinguish the behaviour of U90 in tumour cells, whether there is an equivalent in control cells and whether U90 can be induced without HSV replication. U90 can be immunoprecipitated from radiolabelled human cells which are capable of extended passage in culture, as well as from human tumour cells, rodent tumour cells and cells of different lineages, e.g. epithelial, fibroblastic and lymphoid. Purification of U90 and the subsequent production of monospecific antibodies revealed, by western blotting, a homologue of 90 kDa in control cells. Western blotting shows that HSV can increase the amount of the U90 homologue in these normal cells.
The absence of an immunoprecipitate of the U90 homologue from control cells is a result of the very short half-life (i.e. high turnover) of the protein in these cells (32.9 minutes) as opposed to tumour cells (about 13 hours). Induction of U90 is an early event after HSV infection, occurring in the absence of viral DNA synthesis, but requiring cellular RNA synthesis.
Infection with HSV-2 is not in itself sufficient to convert the homologue of U90 in control cells to a protein with a similar half-life to that of U90 in tumour cells, showing that a function other than HSV infection is involved in raising the half-life of the U90 homologue to tumour cell levels.
Expression of a mitochondrial stress protein in the protozoan parasite Leishmania major
Authors: Searle Susan , McCrossan Maria V. , Smith Deborah F. 
Published: April 2021

ABSTRACT
The DNA sequence has been determined of a gene from Leishmania major that shares sequence identity with members of the eukaryotic heat shock protein (hsp) 70 gene family. The deduced open reading frame for translation shares a number of features common to hsp70 stress proteins, including conserved amino acids implicated in ATP binding and a putative calmodulin-binding site. In addition, the protein has an N-terminal sequence characteristic of a mitochondrial targeting signal. Specific antibodies to this protein, generated by the use of recombinant fusion peptides, recognise a 65 kDa molecule of pI 6.7. This molecule is constitutively expressed and localises to the mitochondrion in all stages of the parasite life cycle. These features suggest a role for this protein as a molecular chaperone in Leishmania.
Formation of the annexin II2p112 complex upon differentiation of F9 teratocarcinoma cells
Authors: Harder Thomas , Thiel Carsten , Gerke Volker 
Published: April 2021

ABSTRACT
Murine teratocarcinoma F9 cells, which remain undifferentiated under standard cell culture conditions, can form cellular layers resembling early embryonic tissues upon induction of differentiation by retinoic acid and cyclic AMP. We have employed a combination of Northern and Western blot analyses to elucidate the regulation of expression of the tyrosine kinase substrate annexin II and its cellular ligand p11 during this differentiation process. Interestingly, the synthesis of the two subunits of the annexin II2p112 complex is not co-regulated during F9 differentiation. Annexin II, which is only very weakly expressed in undifferentiated F9 cells, shows a strong increase in the amount of transcript and protein once the differentiated phenotype is established. The level of this induction does not depend on the type of F9 differentiation. In contrast to the regulated synthesis of annexin II, a significant amount of p11 mRNA and protein is already present in the undifferentiated cells and remains constant during the differentiation of F9 cells. Immunofluorescence analysis reveals that annexin II and p11 are concentrated in the submembranous region of the differentiated F9 cells. In contrast, p11 is uniformly distributed throughout the cytoplasm of undifferentiated cells. p11 is translocated to the submembranous region of the undifferentiated F9 cells upon coexpression of an exogenous annexin II introduced by transient transfection. Thus the localization of annexin II and p11 to the submembranous cytoskeleton depends on the formation of the tight annexin II 2p112 complex.
Calmodulin and the contractile vacuole complex in mitotic cells of Dictyostelium discoideum
Authors: Zhu Qianlong , Liu Tongyao , Clarke Margaret 
Published: April 2021

ABSTRACT
In amoebae of the eukaryotic microorganism Dictyostelium discoideum, calsmodulin is greatly enriched on membranes of the contractile vacuole complex, an osmoregulatory organelle. Antibodies specific for Dictyostelium calmodulin were used in the present study to immunolocalize the contractile vacuole complex in relation to the Golgi complex (detected with wheat germ agglutinin) and the microtubule organizing center (MTOC, detected with anti-tubulin antibodies). Cells were examined throughout the cell cycle. Double-staining experiments indicated that the contractile vacuole complex extended to the MTOC in interphase cells, usually, but not always, overlapping the Golgi complex. In metaphase and anaphase cells, the Golgi staining became diffuse, suggesting dispersal of Golgi membranes. In the same mitotic cells, anti-calmodulin antibodies labeled numerous small cortical vacuoles, indicating that the contractile vacuole complex had also become dispersed. When living mitotic cells were examined, the small cortical vacuoles were seen to be active, implying that all parts of the Dictyostelium contractile vacuole complex possess the ability to accumulate fluid and fuse with the plasma membrane. In contrast to observations reported for other types of cells, anti-calmodulin antibodies did not label the mitotic spindle in Dictyostelium. Despite this difference in localization, it is possible that vacuole-associated calmodulin in Dictyostelium cells and spindle-associated calmodulin in larger eukaryotic cells might perform a similar function, namely, regulating calcium levels.
Effect of shear stress upon localization of the Golgi apparatus and microtubule organizing center in isolated cultured endothelial cells
Authors: Coan Daniel E. , Wechezak Arlene R. , Viggers Robert F. , Sauvage Lester R. 
Published: April 2021

ABSTRACT
Despite substantial evidence to suggest that directed cell migration is dependent upon positioning of the Golgi apparatus (GA) and the microtubule organizing center (MTOC), some controversy exists about whether such a relationship is relevant to endothelial cells under flow. The present study was undertaken to provide an indepth investigation of the relationship between shear stress, GA/MTOC localization, cell migration and nuclear position. Bovine carotid endothelial cells were exposed to 22 or 88 dynes/cm2 for 0.5, 2, 8 or 24 h, and localization of their GA/MTOCs was determined relative to the direction of flow. In no-flow control specimens, (0, 0.5, 2, 8 and 24 h) there was no change in the equally distributed GA/MTOCs. In contrast, during the first 8 h at 88 dynes/cm2 and by 2 h at 22 dynes/cm2 there was a significant increase in the number of cells with GA/MTOCs localized upstream to flow direction. The effect was temporary, however, and by 24 h there was no significant difference between the no-flow, 22 and 88 dynes/cm2 specimens. Analysis of GA/MTOC localization with respect to the direction of cell migration determined that 72.5% of no-flow cells possessed GA/MTOCs localized to the sides of nuclei nearest the direction of migration. In contrast, 64% of the specimens shear stressed over the same time period had GA/MTOCs localized to the sides of nuclei opposite the direction of migration. These results suggest that positioning of the GA/MTOC in endothelial cells is not dependent completely upon the direction of migration. Also, analysis of GA/MTOC localization with respect to nuclear positioning determined that restrictions within the perinuclear space are not solely responsible for GA/MTOC localization.