bluuebunny/crossref_metadata_2025_split huggingface dataset:
The casein kinase 1α gene of Drosophila melanogaster is developmentally regulated and the kinase activity of the protein induced by DNA damage
Authors: Santos João A. , Logarinho Elsa , Tapia Claudio , Allende Catherine C. , Allende Jorge E. , Sunkel Claudio E. 
Published: April 2021

ABSTRACT
We report the molecular cloning and characterisation of the first CK1(casein kinase) gene of Drosophila melanogaster (dmCK1). The protein sequence (DMCK1) shares significant homology with other mammalian CK1 protein kinases of the α sub-class. The dmCK1 gene is expressed only in adult females and during early embryonic development as a single transcript. Western blot analysis of total protein extracts of different stages of development show that the gene product is likewise present during early embryogenesis and in adult females. Kinase activity studies show that DMCK1 is active when in vitro translated but inactive when immunoprecipitated from total early embryo extracts. However, after dephosphorylation treatment the immunoprecipitates show high kinase activity. More significantly, DMCK1 kinase activity present in the immunoprecipitates can be specifically activated by γ-irradiation of early embryos. Also, when DMCK1 is immunoprecipitated after irradiation it appears to undergo phosphorylation. Immunolocalization of DMCK1 in early embryos shows that the protein is predominantly cytoplasmic but after irradiation there is a significant relocalization to the interphase nucleus. The results suggest a possible requirement of the Drosophila CK1α for mechanisms associated with DNA repair during early embryogenesis.
Intracellular redistribution of Ku immunoreactivity in response to cell-cell contact and growth modulating components in the medium
Authors: Fewell Joseph W. , Kuff Edward L. 
Published: April 2021

ABSTRACT
Ku is a heterodimeric protein first recognized as a human autoantigen but now known to be widely distributed in mammalian cells. Analysis of repair-deficient mutant cells has shown that Ku is required for DNA repair, and roles in DNA replication and transcription have also been suggested on the basis of in vitro observations. Ku is generally regarded as a nuclear component. However, in the present paper, we show that a quantitatively significant fraction (half or more) of Ku is located in the cytoplasm of cultured primate cells, and that major changes in epitope accessibility of both nuclear and cytoplasmic Ku components are associated with the transition from sparse to confluent cell densities. The same changes in immunoreactivity were seen in HeLa, 293, CV-1 (monkey) and HPV-transformed keratinocyte cell lines, and in primary cultures of human keratinocytes. The immunostaining pattern of sparsely grown cells could be converted to the ‘confluent’ configuration by re-plating them at the same low density on a monolayer of mouse 3T3 cells. The confluent antigen pattern could also be induced in sparse cells within 15–30 minutes by exposure of the cells to serum- or Ca2+-free medium or overnight with 2 mM hydroxyurea. Somatostatin at 0.12 mM blocked the effects of serum/Ca2+ deprivation on Ku p70 antigen distribution in sparse CV-1 cells, and in confluent cultures reversed the usual nuclear concentration of p70 immunoreactivity. However, somatostatin did not alter the expected immunostaining patterns of p86. Preliminary studies indicate that sparse CV-1 cells, but not HeLa cells, respond to as little as 1 pM of TGF-β1 in the culture medium by the rapid appearance of nuclear immunoreactivity. TGF-α had no apparent effect. These findings are consistent with the participation of Ku in a signal transduction system responsive to the inhibitory effect of cell-cell contact on the one hand and to cytokines and growth-supportive components of the culture medium on the other.
The role of the membrane-spanning domain and stalk region of N-acetylglucosaminyltransferase I in retention, kin recognition and structural maintenance of the Golgi apparatus in HeLa cells
Authors: Nilsson Tommy , Rabouille Catherine , Hui Norman , Watson Rose , Warren Graham 
Published: April 2021

ABSTRACT
Using a series of chimeric and truncated N-acetylglucosaminyltransferase I (NAGT I) molecules we have shown that part of the lumenal stalk region is both necessary and sufficient for kin recognition of mannosidase II and retention in the Golgi stack. The membrane-spanning domain was not required for retention, but replacing part or all of this domain with leucine residues did have a dramatic effect on Golgi morphology. In stable cell lines, stacked cisternae were replaced by tubulo-vesicular clusters containing the mutated NAGT I. The loss of stacked cisternae was proportional to the number of leucines used to replace the membrane-spanning domain.
Role of laminin-1 and TGF-β3 in acinar differentiation of a human submandibular gland cell line (HSG)
Authors: Hoffman Matthew P. , Kibbey Maura C. , Letterio John J. , Kleinman Hynda K. 
Published: April 2021

ABSTRACT
Previous studies show that culturing an immortalized human submandibular gland cell line (HSG) on Matrigel, a basement membrane extract, induces cytodifferentiation. We have further defined this model system and identified factors involved in HSG cell acinar development and cytodifferentiation. Acinar development is marked by cell migration into multi-cellular spherical structures, cell proliferation and apoptosis of the centrally localized cells. In addition, functional differentiation was determined by indirect immunofluorescence and immunoblot analysis for cystatin, a salivary gland acinar cell-specific protein found to be produced by differentiated HSG cells. Matrigel contains multiple extracellular matrix proteins, however, laminin-1 was identified as the major matrix component that induced HSG cell acinar development and cytodifferentiation. Antibodies against specific components of Matrigel and against cell surface adhesion molecules were added to cells in culture to identify components important for HSG cell acinar differentiation. Immunostaining of HSG cell acini identified TGF-β2 and β3 as the predominant isoforms within the cells. Neutralizing antibodies directed against TGF-β3 significantly decreased (P≤0.0002) the size of acini formed. These results indicate that multiple components, including laminin-1 and TGF-β3, contribute to HSG cell acinar development. This model system will be useful to study acinar differentiation and salivary glandspecific protein expression in vitro.
Actin filament bundles are required for microtubule reorientation during growth cone turning to avoid an inhibitory guidance cue
Authors: Challacombe Jean F. , Snow Diane M. , Letourneau Paul C. 
Published: April 2021

ABSTRACT
The extracellular matrix through which growth cones navigate contains molecules, such as chondroitin sulfate proteoglycan, that can inhibit growth cone advance and induce branching and turning. Growth cone turning is accompanied by rearrangement of the cytoskeleton. To identify changes in the organization of actin filaments and microtubules that occur as growth cones turn, we used time-lapse phase contrast videomicroscopy to observe embryonic chick dorsal root ganglion neuronal growth cones at a substratum border between fibronectin and chondroitin sulfate proteoglycan, in the presence and absence of cytochalasin B. Growth cones were fixed and immunocytochemically labeled to identify actin filaments and dynamic and stable microtubules.
Our results suggest that microtubules are rearranged within growth cones to accomplish turning to avoid chondroitin sulfate proteoglycan. Compared to growth cones migrating on fibronectin, turning growth cones were more narrow, and they contained dynamic microtubules that were closer to the leading edge and were more bundled. Cytochalasin B-treated growth cones sidestepped laterally along the border instead of turning, and in sidestepping growth cones, microtubules were not bundled and aligned. We conclude that actin filament bundles are required for microtubule reorientation and growth cone turning to avoid chondroitin sulfate proteoglycan.
Single particle tracking of cell-surface HLA-DR molecules using R-phycoerythrin labeled monoclonal antibodies and fluorescence digital imaging
Authors: Wilson Keith M. , Morrison Ian E. G. , Smith Patricia R. , Fernandez Nelson , Cherry Richard J. 
Published: April 2021

ABSTRACT
The mobility of cell surface MHC molecules and their ability to form dynamic associations may be related to the physiological status of the cell and to the potential to bind effector T lymphocytes. To investigate these properties, we have prepared HLA DR specific monoclonal antibodies coupled in a 1:1 mole ratio to the fluorescent phycobiliprotein, R-phycoerythrin (PE). We show that these small particles can be sequentially imaged using a cooled slow-scan charge coupled device camera and hence can be used for single particle tracking experiments. We have applied this technique to investigate the movements of HLA DR molecules on fibroblasts transfected with human DR alpha and DR beta genes. PE-IgG was bound to the transfected fibroblasts and particle tracks were obtained by sequential imaging over a period of typically 30 minutes. Analysis of particle tracks revealed the presence of directed motion and domain-limited diffusion in addition to random diffusion. The contributions of these three types of motion showed cell to cell variability. Velocities of directed motion were of the order of 2 nm second-1 whilst domain diameters were in the range 200-800 nm. Diffusion coefficients for random diffusion were in the range 1×10−13-5×10−12 cm2 second-1. The higher mobilities were observed for the lower intensity fluorescent spots, which possibly correspond to images of single particles. Much lower mobility was observed with a cell where the spot intensities were approximately double that of the lower intensity spots. These spots could be images of double particles implying the association of at least two HLA DR alpha beta dimers. These data are relevant to the study of MHC class II cell surface redistribution and antigen presentation in specific immunity.
The envelope of vaccinia virus reveals an unusual phospholipid in Golgi complex membranes
Authors: Cluett Edward B. , Machamer Carolyn E. 
Published: April 2021

ABSTRACT
We isolated two forms of enveloped vaccinia virus from infected HeLa cells to obtain membranes for the analysis of lipids of the cis-Golgi network and trans-Golgi network. The intracellular mature virus obtains its envelope by wrapping itself in the membranes of the cis-Golgi network. A fraction of these virions then acquires a second envelope by enwrapping trans-Golgi network membranes to form the intracellular enveloped virus. Lipids were analyzed by high performance thin layer chromatography and digital densitometry to establish a steady-state lipid profile of viral membranes, which should reflect the compositions of the cis-Golgi network and trans-Golgi network. Phosphatidylinositol was slightly enriched in the cis-Golgi network of HeLa cells, whereas the trans-Golgi network showed a minor increase in phosphatidylserine and sphingomyelin. Similarly, cholesterol was only slightly more abundant in the trans-Golgi compared to the cis-Golgi. An unusual lipid, semilysobisphosphatidic acid, was present in significant amounts in vaccinia envelopes. Semilysobisphosphatidic acid was present in similar levels in infected and uninfected cells, and was therefore not induced by vaccinia infection. Subcellular fractionation of HeLa cells indicated that the recovery of semilysobisphosphatidic acid paralleled the recovery of a Golgi marker. Furthermore, a lipid species that comigrated with semilysobisphosphatidic acid was also present in lipids extracted from highly purified, intact Golgi complexes from rat liver. Together, these results suggest that semilysobisphosphatidic acid is a normal component of Golgi membranes.
Contrasting migratory response of astrocytoma cells to tenascin mediated by different integrins
Authors: Giese Alf , Loo Melinda A. , Norman Sylvia A. , Treasurywala Sherri , Berens Michael E. 
Published: April 2021

ABSTRACT
Tenascin, an extracellular matrix protein, is expressed in human gliomas in vitro and in vivo. The distribution of tenascin at the invasive edge of these tumors, even surrounding solitary invading cells, suggests a role for this protein as a regulator of glioma cell migration. We tested whether purified tenascin, passively deposited on surfaces, influenced the adhesion or migration of a human gliomaderived cell line, SF-767. Adhesion of glioma cells to tenascin increased in a dose-dependent fashion up to a coating concentration of 10 µg/ml. Higher coating concentrations resulted in progressively fewer cells attaching. Cell adhesion could be blocked to basal levels using anti-β1 integrin antibodies. In contrast, when anti-αv antibodies were added to the medium of cells on tenascin, cell adhesion was enhanced slightly. Using a microliter scale migration assay, we found that cell motility on tenascin was dose dependently stimulated at coating concentrations of 1 and 3 µg/ml, but migration was inhibited below levels of nonspecific motility when tested at coating concentrations of 30 and 100 µg/ml. Migration on permissive concentrations of tenascin could be reversibly inhibited with anti-β1, while treatment with anti-αv antibodies increased migration rates. We conclude that SF-767 glioma cells express two separate integrin receptors that mediate contrasting adhesive and migratory responses to tenascin.
The fission yeast STS5+ gene is required for maintenance of growth polarity and functionally interacts with protein kinase C and an osmosensing MAP-kinase pathway
Authors: Toda Takashi , Niwa Hajime , Nemoto Takeshi , Dhut Susheela , Eddison Mark , Matsusaka Takahiro , Yanagida Mitsuhiro , Hirata Dai 
Published: April 2021

ABSTRACT
Cell morphogenesis is a fundamental phenomenon that involves understanding a number of biological processes including the developmental program, polarity and cell division. Fission yeast sts5 mutant cells are round rather than cylindrical with cortical actin randomly dispersed. Genetic analyses demonstrate that the sts5+ gene is required for maintenance of cell shape during interphase when the cell normally exhibits polarised growth. The sts5 mutant is not defective in cell wall integrity. Deletion of ppe1+, which encodes a type 2A-like protein phosphatase, shows similar phenotypes to the sts5 mutant and these two mutations are synthetically lethal. Multicopy plasmids containing either the protein kinase C-like gene pck1+ or the protein tyrosine phosphatase pyp1+, an inhibitor of an osmosensing Sty1/Spc1 MAP-kinase, are capable of suppressing the sts5 mutation. Consistent with this, we have found that the wis1 mutation, which is defective in a MAP-kinase kinase of the pathway, suppresses the sts5 mutation. The predicted sts5+ gene product exhibits sequence similarity to two yeast proteins, Dis3 and Ssd1 and a nematode protein, F46E8.6, where the former two yeast proteins have been shown to be involved in cell cycle control and cell morphogenesis. The sts5+ gene is not essential for cell viability, but is absolutely required for polarised growth as the gene disruption showed the same phenotypes as those of the original mutants. Overexpression of the sts5+ gene resulted in altered cell morphology and, cortical actin in these overproducing cells was also abnormal, fainter and often dispersed. Anti-Sts5 antibody specifically detected a 130 kDa protein by western blotting. A green fluorescent protein-Sts5 fusion protein localised in the cytoplasm with a discrete punctate pattern, suggesting that the Sts5 protein is a component of a novel structure. These results have indicated that the Sts5 protein is a crucial determinant of polarised growth and that it functionally interacts with the serine/threonine phosphatase, protein kinase C, and an osmosensing MAP-kinase to maintain cell morphology.
Endo-exonuclease of human leukaemic cells: Evidence for a role in apoptosis
Authors: Fraser Murray J. , Tynan Stephen J. , Papaioannou Arthur , Ireland Christine M. , Pittman Sally M. 
Published: April 2021

ABSTRACT
Inactive forms of endo-exonuclease, activated in vitro by treatment with trypsin, have been identified in human leukaemic CEM and MOLT-4 cells. They comprise over 95% of the total single-strand DNase activity in nuclei and are mainly bound to chromatin and the nuclear matrix. The activated enzyme had Mg2+(Mn2+)-dependent, Ca2+-stimulated activities with single- and double-strand DNAs and RNA (polyriboadenylic acid) and other properties characteristic of endo-exonucleases previously described. At least twice as much inactive endo-exonuclease has also been localised in extranuclear compartments of CEM and MOLT-4 cells, 85% bound to the membranes of the endoplasmic reticulum and 15% free in the cytosol. The soluble cytosolic trypsin-activatable endo-exonuclease was immunoprecipitated by antibodies raised independently to both Neurospora and monkey CV-1 cell endo-exonucleases. The free and bound enzymes of both nuclear and extranuclear compartments also cross-reacted on immunoblots with the antibody raised to Neurospora endo-exonuclease to reveal multiple polypeptides ranging in size from 18 to 145 kDa, many of which exhibited activity on DNA gels. The major species bound to the chromatin/matrix were in the 55-63 kDa range. Limited proteolysis of the large polypeptides to those of 18 to 46 kDa accompanied spontaneous chromatin DNA fragmentation to form DNA ‘ladders’ in an isolated nuclei/cytosol system.
When the leukaemic cells were treated in culture with either etoposide or podophyllotoxin to induce apoptosis, the largest polypeptides disappeared and smaller endo-exonuclease-related polypeptides of 18 to 46 kDa were detected in the nuclear extracts. The appearance of these polypeptides also correlated with extensive chromatin DNA fragmentation. In addition, there were correlations between the depletion of the major 55-63 kDa species bound to the membranes of the endoplasmic reticulum, depletion of the extranuclear trypsin-activatable activity and the onset and extent of chromatin DNA fragmentation in both cell lines. The extranuclear 55-63 kDa species may be precursors of the chromatin/matrix bound endo-exonuclease. The results indicate that endo-exonuclease plays a role in chromatin DNA degradation in mammalian cells during apoptosis.
Quality control in protein biogenesis: Thiol-mediated retention monitors the redox state of proteins in the endoplasmic reticulum
Authors: Brewer Joseph W. , Corley Ronald B. 
Published: April 2021

ABSTRACT
There is accumulating evidence that proteins can be retained in the endoplasmic reticulum by a mechanism that is believed to monitor the oxidation status of one or more cysteines in their sequences. For example, a single cysteine residue critical for retention of secretory IgM assembly intermediates has been mapped to the C-terminal cysteine, Cys575, of the secretory µ chain. Little is known concerning the mechanism responsible for this system of quality control, which has been termed thiolmediated retention. In particular, it is not known if the mechanism monitors the redox state of the important cysteine residue in the secretory µ protein itself or within the context of higher-order IgM complexes. To address this question, we evaluated the fidelity of retention of secretory IgM and determined the redox status of cysteines in secretory µ proteins in polymers and polymer intermediates at various stages of maturation. We demonstrate that all secreting B cells and B cell lines secrete assembly intermediates in addition to completed, covalent pentameric and hexameric IgM polymers. A fraction of assembly intermediates exit the endoplasmic reticulum as individual components, mature through the Golgi without undergoing further assembly, and most, if not all, are secreted. While the majority of IgM assembly intermediates have exposed thiols and are contained within the endoplasmic reticulum where they can be utilized for oligomerization, maturing assembly intermediates found in the Golgi and extracellular space are completely oxidized. Thus, while the retention of unpolymerized IgM is highly efficient, the retention system lacks the ability to distinguish fully oxidized assembly intermediates from fully oxidized completed polymers. The molecular mechanisms that may contribute to this aspect of IgM biogenesis and their implications for the concept of thiolmediated retention are discussed.
Transport of protein kinase C α into the nucleus requires intact cytoskeleton while the transport of a protein containing a canonical nuclear localization signal does not
Authors: Schmalz Dirk , Kalkbrenner Frank , Hucho Ferdinand , Buchner Klaus 
Published: April 2021

ABSTRACT
Protein kinase C undergoes a redistribution from the cytosol into the nucleus upon various stimuli. Since protein kinase C does not contain any known nuclear localization signal, the exact pathway and mechanism of the translocation into the nucleus is not known.
We used immunofluorescence microscopy to investigate the role of the cytoskeleton in this process, and to detect the subcellular distribution of protein kinase C α in NIH 3T3 fibroblasts. In these cells protein kinase C α is translocated into the nucleus after stimulation with phorbol ester. We observed that cells treated with the cytoskeleton disrupting agents cytochalasin B or colchicine do not show the nuclear translocation of protein kinase C α after stimulation. In contrast, the nuclear accumulation of a nuclear localization signal containing reporter protein in an in vitro nuclear transport assay is not affected by these drugs. This observation has been confirmed for intact cells by microinjection experiments: cells which have been incubated with cytochalasin B or colchicine prior to microinjection of the reporter protein show the same accumulation in the nucleus as untreated cells.
Our data show that intact cytoskeleton plays an important role in the translocation of protein kinase C α into the nucleus but not in the nuclear import of a karyophilic reporter protein.
Regulation of the acquisition of meiotic competence in the mouse: Changes in the subcellular localization of cdc2, cyclin B1, cdc25C and weel, and in the concentration of these proteins and their transcripts
Authors: Mitra Jayashree , Schultz Richard M. 
Published: April 2021

ABSTRACT
During their development, mammalian oocytes acquire the ability to resume meiosis. We demonstrate that the concentration of p34cdc2 increases during the acquisition of meiotic competence, as determined by immunoblotting, whereas the concentration of cyclin B1 decreases. Laserscanning confocal microscopy corroborated these changes and furthermore indicate that an increase occurs in the nuclear concentration of each protein. Results of immunoblotting experiments demonstrate that associated with the acquisition of meiotic competence is an increase in the concentration of cdc25C, an activator of p34cdc2/cyclin B kinase, and a decrease in wee1, an inhibitor of cdc2/cyclin B kinase. These changes were again corroborated by laserscanning confocal microscopy, which also indicates that an increase in the nuclear concentration of wee1 occurs. The concentration of the transcripts encoding these proteins, however, is essentially similar in meiotically incompetent and competent oocytes. Thus, these changes in protein concentration that occur during oocyte development likely reflect changes in the translational efficiency of their mRNAs. Consistent with this is that the relative rate of synthesis of p34cdc2 in meiotically competent oocytes is ∼3 times greater than that in meiotically incompetent oocytes, whereas the stability of newly synthesized p34cdc2 is essentially the same in each cell type.