bluuebunny/crossref_metadata_2025_split huggingface dataset:
Contrasting effects of K8 and K18 on stabilizing K19 expression, cell motility and tumorigenicity in the BSp73 adenocarcinoma
Authors: Pankov Roumen , Simcha Inbal , Zöller Margot , Oshima Robert G. , Ben-Ze’ev Avri 
Published: April 2021

ABSTRACT
The co-expression of vimentin and keratin-type intermediate filaments in the same cell was often reported to correlate with increased invasiveness and a more aggressive tumorigenic phenotype. To address the possible physiological relevance of these observations, we transfected simple keratins (K8 and 18) either individually, or in combination, into a tumorigenic but non-metastatic pancreatic adenocarcinoma that expresses vimentin but no keratins. Expression of K8 resulted in the stabilization of endogenous K19 in these cells, and formation of keratin filaments containing K8 and K19. Transfection of K18 yielded unstable K18 protein, but K18 could be stabilized when K8 was co-expressed in the same cells. Clones expressing K18 alone, or together with K8, displayed a reduced ability to grow in soft agar and decreased motility when compared to control, or K8/19 expressing cells. Moreover, K18 expressing cells were dramatically inhibited in their ability to form tumors when injected into syngeneic animals. The extent of suppression in the tumorigenicity of these cells correlated with the level of K18 expressed by these cells. The results show that K18 expression in cells may result in the suppression of the motile and tumorigenic abilities of this adenocarcinoma.
Activation of the β2 integrin Mac-1 (CD11b/CD18) by an endogenous lipid mediator of human neutrophils and HL-60 cells
Authors: Klugewitz Katja , Ley Klaus , Schuppan Detlef , Nuck Rolf , Gaehtgens Peter , Walzog Barbara 
Published: April 2021

ABSTRACT
β2 integrins (CD11/CD18) play a key role in the adhesion, activation, migration and phagocytosis of human neutrophils. In order to exert their functions, β2 integrins require activation, which results in an enhancement of ligand affinity. This functional up-regulation is probably due to a conformational change of the β2 integrins, but the mechanisms of inside-out signaling that trigger this activation are still under investigation. In the present study, the effect of cellular lipids on the affinity state of β2 integrins was investigated. Lipids were extracted from human neutrophils and HL-60 cells after stimulation with IL-8 or phorbol ester, respectively. The extracts were purified by anion exchange chromatography and/or HPLC fractionation. The lipid extracts induced the adhesion of neutrophils to fibrinogen and, in a cell-free assay system, the binding of C3bi-coated zymosan-particles by purified β2 integrin Mac-1 (CD11b/CD18). The integrin up-regulating activity was resistant to ester hydrolysis, eluted as one particular HPLC-fraction, and showed an absorption maximum at 194±2 nm. Taken together, these data support the concept that activated neutrophils and HL-60 cells can generate an endogenous lipid mediator, which up-regulates ligand binding activity of β2 integrins.
AND-1, a natural chimeric DNA-binding protein, combines an HMG-box with regulatory WD-repeats
Authors: Köhler Andreas , Schmidt-Zachmann Marion S. , Franke Werner W. 
Published: April 2021

ABSTRACT
Using a specific monoclonal antibody (mAb AND-1/23-5-14) we have identified, cDNA-cloned and characterized a novel DNA-binding protein of the clawed toad, Xenopus laevis, that is accumulated in the nucleoplasm of oocytes and various other cells. This protein comprises 1,127 amino acids, with a total molecular mass of 125 kDa and a pI of 5.27. It is encoded by a mRNA of ∼4 kb and contains, in addition to clusters of acidic amino acids, two hallmark motifs: the amino-terminal part harbours seven consecutive ‘WD-repeats’, which are sequence motifs of about 40 amino acids that are characteristic of a large group of regulatory proteins involved in diverse cellular functions, while the carboxy terminal portion possesses a 63-amino-acid-long ‘HMG-box’, which is typical of a family of DNA-binding proteins involved in regulation of chromatin assembly, transcription and replication. The DNA-binding capability of the protein was demonstrated by DNA affinity chromatography and electrophoretic mobility shift assays using four-way junction DNA. Protein AND-1 (acidic nucleoplasmic DNA-binding protein) appears as an oligomer, probably a homodimer, and has been localized throughout the entire interchromatinic space of the interphase nucleoplasm, whereas during mitosis it is transiently dispersed over the cytoplasm. We also identified a closely related, perhaps orthologous protein in mammals. The unique features of protein AND-1, which is a ‘natural chimera’ combining properties of the WD-repeat and the HMG-box families of proteins, are discussed in relation to its possible nuclear functions.
Analysis of Toxoplasma gondii stably transfected with a transmembrane variant of its major surface protein, SAG1
Authors: Seeber Frank , Dubremetz Jean-François , Boothroyd John C. 
Published: April 2021

ABSTRACT
We have genetically engineered Toxoplasma gondii so that its major surface antigen SAG1 is anchored by a human transmembrane domain (SAG1-TM) instead of its natural GPI anchor (SAG1-GPI) in order to initiate studies to address the function of this protein anchor in parasitic protozoa as well as to get insights into the functional role of SAG1. Our results show that SAG1-TM is correctly folded (at least as judged by the presence of conformationally dependent epitopes) and targeted to the surface of the parasite, indicating that the GPI anchor does not determine its localization nor overall three-dimensional structure. No significant difference was seen in any aspect of the growth of the SAG1-TM mutant. However, compared to the natural SAG1-GPI, SAG1-TM does not form strong associations with itself and/or other molecules in high molecular weight complexes suggesting that allowing such complexes to form may be one role of the GPI anchor. The in vitro half-life of SAG1-TM of extracellular parasites is significantly lower than that of SAG1-GPI suggesting a stabilizing function of the glycolipid anchor against degradation and/or membrane release. Antibodies to SAG1 are shed from SAG1-TM parasites as they invade, just as they are stripped from SAG1-GPI bearing parasites. The stripping, therefore, is unlikely to be driven by the action of lipases.
A quantitative analysis of connexin-specific permeability differences of gap junctions expressed in HeLa transfectants and Xenopus oocytes
Authors: Cao Fengli , Eckert Reiner , Elfgang Claudia , Nitsche Johannes M. , Snyder Scott A. , Hülser Dieter F. , Willecke Klaus , Nicholson Bruce J. 
Published: April 2021

ABSTRACT
Gap junctions provide direct intercellular communication by linking adjacent cells with aqueous pores permeable to molecules up to 1 kDa in molecular mass and 8-14 Å in diameter. The identification of over a dozen connexins in the mammalian gap junction family has stimulated interest in the functional significance of this diversity, including the possibility of selectivity for permeants as seen in other channel classes. Here we present a quantitative comparison of channel permeabilities of different connexins expressed in both HeLa transfectants (rat Cx26, rat Cx32 and mouse Cx45) and Xenopus oocytes (rat Cx26 and rat Cx32). In HeLa cells, we examined permeability to two fluorescent molecules: Lucifer Yellow (LY: anionic, MW 457) and 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI, cationic, MW 350). A comparison of the kinetics of fluorescent dye transfer showed Cx32, Cx26 and Cx45 to have progressively decreasing permeabilities to LY, but increasing permeabilities to DAPI. This pattern was inconsistent with selection based on physical size of the probe, nor could it be accounted for by the differences between clones in the electrical conductance of the monolayers. In Xenopus oocytes, where electrical and dye coupling could be assessed in the same cells, Cx32 coupled oocytes showed an estimated 6-fold greater permeability to LY than those coupled by Cx26, a comparable result to that seen in HeLa cells, where an approximately 9-fold difference was seen. The oocyte system also allowed an examination of Cx32/Cx26 heterotypic gap junction that proved to have a permeability intermediate between the two homotypic forms. Thus, independent of the expression system, it appears that connexins show differential permeabilities that cannot be predicted based on size considerations, but must depend on other features of the probe, such as charge.
Identification of intracellular sites of superoxide production in stimulated neutrophils
Authors: Kobayashi Toshihiro , Robinson John M. , Seguchi Harumichi 
Published: April 2021

ABSTRACT
In this study, we show that superoxide production is carried out within intracellular compartments of human neutrophils and not at the plasma membrane following stimulation with phorbol myristate acetate. Oxidant production was not observed in unstimulated cells. Stimulated cells exhibited superoxide production in two distinct types of intracellular organelles. Initially, activity was detected in slender rod-shaped granules and in spherical or elliptical granules. The oxidant-producing granules fused directly with the plasma membrane or fused to form larger intracellular vesicles which then became associated with the plasma membrane. Longer periods of stimulation with PMA resulted in a decrease in the number of vesicles containing oxidant reaction product only, and an increase in structures containing both the oxidant-reaction product and ferritin particles; the latter was used herein as a marker for endocytosis. Thus a complex pattern of intracellular vesicular trafficking occurs in stimulated neutrophils. Alkaline phosphatase activity, a marker enzyme for a type of intracellular neutrophil granule was co-localized in the oxidant reaction-positive intracellular compartments. The time course of up-regulation of alkaline phosphatase activity to the cell surface parallelled the release of superoxide from stimulated cells. Results from this study demonstrate for the first time cytochemical and morphological evidence that superoxide is released from stimulated neutrophils through exocytosis of an oxidant-producing intracellular granule.
Unidirectional movement of fluorescent microtubules on rows of dynein arms of disintegrated axonemes
Authors: Yamada Akira , Yamaga Takako , Sakakibara Hitoshi , Nakayama Haruto , Oiwa Kazuhiro 
Published: April 2021

ABSTRACT
Tetramethylrhodamine-labelled microtubules were observed to move on rows of dynein arms of sea urchin sperm axonemes exposed by elastase-induced sliding disintegration. The microtubules moved towards the flagellar tip at a velocity of 3.1±2.1 μm second−1 (mean ± s.d., n=53) in the presence of 0.1 mM ATP at 22°C, but none moved towards the sperm head. We also examined the polarity of microtubule binding to axonemal doublet microtubules in the absence of ATP by using microtubules brightly labelled at their minus-ends. In 140 of 210 microtubules studied, they bound to axonemal microtubules with a parallel polarity. These results suggest that tightly packed dynein arms on the outer doublet microtubules of sperm axoneme preferentially bind microtubules to themselves with the same polarity as that of the axoneme and that they generate a force to move only these microtubules in the direction away from the sperm head.
Protein phosphatase 4 is an essential enzyme required for organisation of microtubules at centrosomes in Drosophila embryos
Authors: Helps Nicholas R. , Brewis Neil D. , Lineruth Katrin , Davis Terence , Kaiser Kim , Cohen Patricia T. W. 
Published: April 2021

ABSTRACT
The protein serine/threonine phosphatase 4 (PP4), which localises to centrosomes/spindle pole bodies in human cells, is shown to exhibit a similar localisation in Drosophila cells and embryos and possess a highly conserved (91% identical) amino acid sequence from humans to invertebrates. A homozygous Drosophila melanogaster strain mutant in the PP4 gene at 19C1-2 has been produced using P element mutagenesis. This strain, termed centrosomes minus microtubules (cmm), has reduced amounts of PP4 mRNA, ∼25% of normal PP4 protein in early embryos and exhibits a semi-lethal phenotype with only 10% viability in certain conditions. Reversion mutagenesis shows that the phenotype is due to the presence of the P element in the PP4 mRNA. In early cmm embryos, nuclear divisions become asynchronous and large regions containing centrosomes with no well defined radiating microtubules are visible. In such areas, most nuclei arrest during mitosis with condensed DNA, and mitotic spindle microtubules are either absent, or aberrant and unconnected to the centrosome. A reduction in the staining of γ-tubulin at centrosomes in cmm embryos suggests a conformational change or relocation of this protein, which is known to be essential for initiation of microtubule growth. These findings indicate that PP4 is required for nucleation, growth and/or stabilisation of microtubules at centrosomes/spindle pole bodies.
Lipocortin 1 (annexin 1) in patches associated with the membrane of a lung adenocarcinoma cell line and in the cell cytoplasm
Authors: Traverso Valerie , Morris John F. , Flower Rod J. , Buckingham Julia 
Published: April 2021

ABSTRACT
Lipocortin 1 (annexin I) is a calcium- and phospholipidbinding annexin protein which can be externalised from cells despite the lack of a signal sequence. To determine its cellular distribution lipocortin 1 in A549 human lung adenocarcinoma cells was localised by light- and electron-microscopic immunocytochemistry and by cell fractionation and western blotting. Lipocortin 1 immunoreactivity is concentrated in prominent patches associated with the plasma membrane. The intensity of these patches varied with the confluence and duration of the culture and was not detectably diminished by an EDTA wash before fixation. Tubulin and cytokeratin 8 were colocalized with lipocortin 1 in the patches. Within the cells lipocortin 1 was distributed throughout the cytoplasm. Electron microscopy revealed prominent immunoreactivity along the plasma membrane with occasional large clusters of gold particles in contact with the membrane surface of the cells; within the cytoplasm the membrane of some vesicle/vacuole structures and some small electron-dense bodies was immunoreactive, but no immunogold particles were associated with the multilamellar bodies. Subcellular fractionation, extraction and western blotting showed that lipocortin 1 in the membrane pellet was present as two distinct fractions; one, intimately associated with the lipid bilayer, which behaved like an integral membrane protein and one loosely attached which behaved like a peripheral membrane protein. The results show that a substantial amounts of lipocortin 1 is concentrated in focal structures associated with and immediately beneath the plasma membrane. These might form part of the mechanism by which lipocortin 1 is released from the cells.
Sequential silver staining and in situ hybridization reveal a direct association between rDNA levels and the expression of homologous nucleolar organizing regions: a hypothesis for NOR structure and function
Authors: Zurita F. , Jiménez R. , Burgos M. , Díaz de la Guardia R. 
Published: April 2021

ABSTRACT
We have developed a procedure for sequential silver staining and in situ hybridization to analyze the relationship between the amount of rDNA present in nucleolar organizer regions, as estimated by in situ hybridization, and their level of expression, as estimated by the silver signal. For simplicity we used cells from the insectivorous mole Talpa occidentals, which have a single pair of nucleolar organizer regions in chromosome pair 3. The relative content of ribosomal cistrons was also related to the hierarchy of activation of the nucleolar organizer regions present in this chromosomal pair. Statistical analyses demonstrated that both the relative level of expression and the activation hierarchy depended mainly on the number of ribosomal cistrons in nucleolar organizer regions. We propose a functional two-step hypothesis, which is consistent with most known data concerning interchromosomal, intercellular and interindividual variation in a number of plant and animal species, including Talpa occidentals. In step one, the first available transcription factors bind randomly to the ribosomal promoters, such that larger nucleolar organizer regions are more likely to recruit them. In the second step the remaining transcription factors are recruited in a cooperative way, thus completing activation of one nucleolar organizer region, before the next one becomes active.
Direct cloning and analysis of DNA sequences from a region of the Chinese hamster genome associated with aphidicolin-sensitive fragility
Authors: Palin Andrew H. , Critcher Ricky , Fitzgerald Daniel J. , Anderson John N. , Farr Christine J. 
Published: April 2021

ABSTRACT
Fragile sites are reproducibly expressed and chemically induced decondensations on mitotic chromosomes observed under cytological conditions. They are classified both on the basis of the frequency with which they occur (rare and common) and in terms of the chemical agent used to induce expression in tissue culture cells. Aphidicolin-sensitive common fragile sites appear to be ubiquitous in humans and other mammals and have been considered as candidates of pathological importance. Recently DNA from FRA3B, the most highly expressed constitutive fragile site in the human genome, has been cloned although as yet the cause of the underlying fragility has not been identified. In this study we describe the isolation, using a direct cloning approach, of DNA from a region of the Chinese hamster genome associated with aphidicolin-inducible fragility. Cells of a human-hamster somatic cell hybrid were transfected with a pSV2HPRT vector while exposed to aphidicolin, an inhibitor of DNA polymerases α, δ and ε. FISH analysis of stable transfectant clones revealed that the ingoing plasmid DNA had preferentially integrated into fragile site-containing chromosomal bands. Plasmid rescue was used to recover DNA sequences flanking one such integration site in the hamster genome. We demonstrate by FISH analysis of metaphase cells induced with aphidicolin that the rescued DNA is from a region of fragility on Chinese hamster chromosome 2, distal to the DHFR locus. Analysis of the DNA sequences flanking the integration site revealed the overall A+T content of the 3,725 bp region sequenced to be 63.3%, with a highly [A]·[T]-RICH 156 bp region (86.5%) almost adjacent to the integration site. Computational analyses have identified strong homologies to Saccharomyces cerevisiae autonomous replicating sequences (ARS), polypyrimidine tracts, scaffold attachment site consensus sequences and a 24 bp consensus sequence highly conserved in eukaryotic replication origins, all of which appear to cluster around the [A]·[T]-rich sequences. This domain also possesses structural characteristics which are common to both prokaryotic and eukaryotic origins of replications, in particular an unusually straight conformation of low thermal stability flanked either side by highly bent DNA segments. Further isolation and characterisation of DNA sequences from common fragile sites will facilitate studies into the underlying nature of these enigmatic regions of the mammalian genome, leading to a greater understanding of chromatin structure.
Actin dynamics in living mammalian cells
Authors: Ballestrem Christoph , Wehrle-Haller Bernhard , Imhof Beat A. 
Published: April 2021

ABSTRACT
The actin cytoskeleton maintains the cellular architecture and mediates cell movements. To explore actin cytoskeletal dynamics, the enhanced green fluorescent protein (EGFP) was fused to human β-actin. The fusion protein was incorporated into actin fibers which became depolymerized upon cytochalasin B treatment. This functional EGFP-actin construct enabled observation of the actin cytoskeleton in living cells by time lapse fluorescence microscopy. Stable expression of the construct was obtained in mammalian cell lines of different tissue origins. In stationary cells, actin rich, ring-like structured ‘actin clouds’ were observed in addition to stress fibers. These ruffle-like structures were found to be involved in the reorganization of the actin cytoskeleton. In migratory cells, EGFP-actin was found in the advancing lamellipodium. Immobile actin spots developed in the lamellipodium and thin actin fibers formed parallel to the leading edge. Thus EGFP-actin expressed in living cells unveiled structures involved in the dynamics of the actin cytoskeleton.
The small GTP-binding protein RacG regulates uroid formation in the protozoan parasite Entamoeba histolytica
Authors: Guillén Nancy , Boquet Patrice , Sansonetti Philippe 
Published: April 2021

ABSTRACT
Entamoeba histolytica is a protozoan parasite that invades human intestine leading to ulceration and destruction of tissue. Amoebic movement and phagocytosis of human cells is accompanied by characteristic changes in cell morphology. Amoebae become polarized, developing a frontal pseudopod and a well-defined rear zone of membrane accumulation designated the uroid. In motile eukaryotic cells, a phenomenon that contributes to movement is the capping of receptors at the cell surface. During the capping process, E. histolytica concentrates ligand-receptor complexes in the uroid. Interestingly, some of these surface receptors are involved in the survival of the parasite. While looking for regulators of capping and uroid formation, we identified RacG, an E. histolytica protein that is homologous to human Rac1. This protein belongs to the Rac subfamily of small GTPases implicated in interactions between the actin cytoskeleton and the membrane of mammalian cells. Cloning of the EhracG gene and analysis of the protein activity either in murine fibroblasts or in E. histolytica revealed that EhRacG induces a characteristic Rac phenotype. When expressed in amoebae, an EhRacG-V12 mutant protein not only deregulated cell polarity, but also caused a defect in cytokinesis. Analysis of the cytoskeleton in amoebae bearing this mutant revealed that F-actin concentrated at the periphery of the cell. In addition, the number and localization of uroids were modified. These results suggest a role for EhRacG in amoebic morphogenesis and cytokinesis.
The ’ ligand-induced conformational change’ of α5βl integrin: Relocation of α5 subunit to uncover the β1 stalk region
Authors: Tsuchida Jun , Ueki Shoko , Takada Yoshikazu , Saito Yuji , Takagi Junichi 
Published: April 2021

ABSTRACT
Integrin heterodimers undergo a conformational change upon the binding of ligand to their extracellular domains. An anti-β1 integrin monoclonal antibody AG89 can detect such a conformational change since it recognizes a ligand-inducible epitope in the stalk-like region of β1 subunits. The binding of a 125I-labeled AG89 Fab fragment to α5β1 integrins on K562 cells was assessed and analyzed by the Scatchard method. High affinity binding sites for AG89 are present on cells treated with ligand peptide. In addition, results revealed that cells treated with EDTA also express AG89 binding sites with the same affinity although the number of binding sites is 4-fold lower. AG89 immunoprecipitated α5β1 complexes from surface-labeled K562 cells treated with ligand peptide. By contrast, it immunoprecipitated only β1 chains when the ligand peptide was absent, suggesting that high affinity binding sites on EDTA-treated cells are associated with non-functional β1 monomer. Additional studies show that the epitope for AG89 is constitutively exposed on mutant β1 that cannot complex with α5. These data suggest that the AG89 epitope is masked by the α5 subunit. Ligand binding and integrin activation may uncover the β1 stalk region by triggering a conformational shift of α5 relative to β1.