bluuebunny/crossref_metadata_2025_split huggingface dataset:
Origins of the parasitophorous vacuole membrane of the malaria parasite, Plasmodium falciparum, in human red blood cells
Authors: Dluzewski A. R. , Mitchell G. H. , Fryer P. R. , Griffiths S. , Wilson R. J. M. , Gratzer W. B. 
Published: April 2021

ABSTRACT
We have attempted to determine whether the parasitophorous vacuole membrane, in which the malaria parasite (merozoite) encapsulates itself when it enters a red blood cell, is derived from the host cell plasma membrane, as the appearance of the invasion process in the electron microscope has been taken to suggest, or from lipid material stored in the merozoite. We have incorporated into the red cell membrane a haptenic phospholipid, phosphatidylethanolamine, containing an NBD (N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)) group, substituted in the acyl chain, and allowed it to translocate into the inner bilayer leaflet. After invasion of these labelled cells by the parasite, Plasmodium falciparum, immuno-gold electron microscopy was used to follow the distribution of the labelled lipid; this was found to be overwhelmingly in favour of the host cell membrane relative to the parasitophorous vacuole. Merozoites of P. knowlesi were allowed to attach irreversibly to red cells without invasion, using the method of pretreatment with cytochalasin. The region of contact between the merozoite and the host cell membrane was in all cases devoid of the labelled phosphatidylethanolamine. These results lead us to infer that the parasitophorous vacuole membrane is derived wholly or partly from lipid preexisting in the merozoite.
Syringe loading introduces macromolecules into living mammalian cell cytosol
Authors: Clarke Mark S. F. , McNeil Paul L. 
Published: April 2021

ABSTRACT
We describe a simple, efficient, gentle and inexpensive technique for the introduction of normally impermeant macromolecules into the cytosol of living mammalian cells growing in suspension or attached to the culturing substratum. Loading is achieved by the production of transient, survivable plasma membrane disruptions as cells are passed back and forth through a standard syringe needle or similar narrow orifice. The loading volume required, which contains cells and the macromolecule to be loaded, can be as little as 5 μl, thus minimizing the use of valuable reagents. In addition, we report that the surfactant molecule, Pluronic F-68, is capable of altering the physical properties of the plasma membrane in such a way as to increase loading efficiency and the long-term survivability of cells loaded by this and other mechanically based cell-loading techniques.
Conditions for assembly of tubulin-based structures in unfertilized sea urchin eggs Spirals, monasters and cytasters
Authors: Harris Patricia J. , Clason Elise L. 
Published: April 2021

ABSTRACT
Cytasters were induced in the unfertilized eggs of the sea urchin Lytechinus pictus by two different methods: (1) treatment with hexylene glycol or taxol, which are known to lower the tubulin critical concentration in vitro, but do not activate the nuclear cycle, and (2) by raising the cytoplasmic pH from that of unfertilized cytoplasm to that of fertilized, which activated the nuclear cycle and initiated tubulin polymerization. In the unactivated eggs, with increasing concentrations of the inducing agents, temperature and duration of treatment, microtubule structures that formed showed a progression from loose microtubule networks and spiral arrays to tightly organized cytasters. In eggs incubated in sea water containing 2.5 or 10 mM ammonium acetate titrated with HC1 or NaOH in steps from pH 5.0 to pH 9.0, the nuclear cycle and tubulin polymerization were initiated at about pH 7.0. The degree of development attainable after three hours was dependent on the pH, with spirals forming at the threshold level of pH 7.0, monasters at PH 7.5, and at PH 8.5 cells formed cytasters, multipolar spindles and even completed multipolar divisions. In unactivated eggs, tubulin was made available by lowering of the tubulin critical concentration; in activated eggs it was made available from some previously unavailable store. The evidence suggests that the amount of assembly-competent tubulin available, regardless of how it is made available, determines the type of structure that is formed, with microtubule bundles and spirals at the lower concentrations and functional cytasters at the upper. We also describe some details of cytaster formation, including the role of microtubule interactions and the movement of dense granules to the aster centers in hexylene glycol-treated eggs.
Structure of the cortical cytoskeleton in mammalian outer hair cells
Authors: Holley M. C. , Kalinec F. , Kachar B. 
Published: April 2021

ABSTRACT
The cortical cytoskeletal lattice in outer hair cells is a two-dimensional actin-based structure, which can be labelled with rhodamine/phalloidin and disrupted by the enzyme deoxyribonuclease I. Structural information from thin sectioned, freeze-etched and negatively stained preparations shows that it is based upon two types of filament that form a cross-linked lattice of circumferential filaments. The cross-links are 70-80 nm long. Measurements of the spacing between circumferential filaments suggest that the lattice is stiffer circumferen-tially than it is longitudinally. Analysis of the orientation of circumferential filaments shows that it is composed of discrete domains of up to 10 μm2. Relative movements between domains could allow substantial changes of cell shape without disrupting the unit structure of the lattice, thus allowing the cell cortex to retain its elastic responses to high-frequency deformations.
Stage-specific tyrosine phosphorylation of actin in Dictyostelium discoideum cells
Authors: Schweiger Anton , Mihalache Oana , Ecke Maria , Gerisch Günther 
Published: April 2021

ABSTRACT
A 45 kDa protein in Dictyostelium discoideum cells that was recognized by a phosphotyrosine-specific antibody was identified by its binding activity to DNase I and its 2D-electrophoretic behavior as actin. The reactivity of actin with the antibody was transiently enhanced for about 30 minutes shortly after starving cells were reintroduced into nutrient medium. This effect indicates a modification of actin that is regulated under physiological conditions. A similar effect was obtained when growing cells were treated with phenylarsine oxide (PAO), an inhibitor of phosphotyrosine phosphatases. This effect was reversed and the cells fully recovered upon addition of the PAO antagonist 2,3-dimercapto-propanol. Starved cells did not show this enhancement of antibody labelling, which indicates that the response to PAO depends on the developmental stage. Phosphorylated amino acid residues were identified after in vivo labelling with [32P]phosphate in the presence of PAO. Part of the radioactivity in the actin band was recovered as phosphotyrosine, another part as phosphoserine.
PAO caused the cells to form elongated blebs, to round up and finally to become immobilized. Fluorescence labelling with phalloidin of cells that were fixed at different times of PAO treatment revealed a progressive decrease in the staining for actin filaments and showed that these alterations in cytoskeleton organization were readily reversible, in accordance with the reversal of tyrosine phosphorylation at actin.
Formation of highly organized skeletal muscle fibers in vitro Comparison with muscle development in vivo
Authors: Swasdison Somporn , Mayne Richard 
Published: April 2021

ABSTRACT
Two methods were developed in which long-term cultures of quail skeletal muscle were established so that all of the muscle fibers develop in a highly oriented manner. The muscle fibers became spontaneously and vigorously contractile and established strong connections with the extracellular matrix at their ends that closely duplicate the structure of the myotendinous junction. A continuous basal lamina was formed around each muscle fiber that contained type IV collagen, laminin and heparan sulfate proteoglycan. With one of the methods, an extensive extracellular matrix developed around each muscle fiber that was highly organized with the formation of a distinctive épimysium, perimysium and endomysium. Analysis of the cultures by both methods for different isoforms of myosin showed expression of an adult form of myosin by some of the muscle cells. The results therefore demonstrate that muscle development in the present culture systems proceeds extensively for several weeks. It will now be possible to investigate directly the structure of the connections between muscle fibers and the extracellular matrix.
Formation of skeletal muscle in vivo from the mouse C2 cell line
Authors: Morgan Jennifer E. , Moore Stephen E. , Walsh Frank S. , Partridge Terence A. 
Published: April 2021

ABSTRACT
The C2 muscle cell line is myogenic in vitro and has been extensively used in studies of muscle cell differentiation. Here, we have investigated the myogenicity in vivo of C2 cells implanted into suitable sites in the mouse.
Large amounts of new muscle were formed when C2 cells were implanted into sites in nude mice which were undergoing regeneration following whole muscle grafting and in scaffolding of freeze-killed muscle or vicryl suture in the anterior tibial compartment. When implanted into regenerating muscle, C2 cells fused with the host muscle to form mosaic fibres; when implanted into inert sites, they formed muscle of largely donor origin. C2-derived muscle fibres appeared to become innervated, but the progression of N-CAM (neural cell adhesion molecule) isoform changes in such regenerates indicated that they did not become fully mature. Proliferating, undifferentiated cells of C2 origin form tumours in older grafts; however, this was more pronounced in the absence of competition from host muscle cells.
In the short term, C2 cells can form large amounts of muscle in vivo for biochemical analysis. In addition, C2 cells are easily manipulable in vitro; genes of interest may be transfected into them prior to implantation of the cells into skeletal muscle and the effects of these genes in vivo may thus be examined.
Total inhibition of involucrin synthesis by a novel two-step antisense procedure Further examination of the relationship between differentiation and malignancy in hybrid cells
Authors: Griffin Elaine F. , Harris Henry 
Published: April 2021

ABSTRACT
A novel procedure involving the sequential use of two different antisense constructs has been used to inhibit the synthesis of involucrin in a hybrid cell line formed by the fusion of a human cervical carcinoma cell with a normal human keratinocyte (ESH100P6). In this cell line, and other similar hybrids, malignancy, as measured by progressive growth in vivo, is suppressed; and it has been shown that the keratinocyte imposes its own programme of terminal differentiation on the non-malignant hybrid cell. In particular, involucrin, a precursor of one of the major components of the cornified envelope of mature kératinocytes, continues to be produced. When, however, malignant segregants arise in the hybrid cell population, the terminal differentiation programme of the keratinocyte is not expressed and involucrin ceases to be made. It seemed possible that if the synthesis of involucrin, a critical marker of keratinocyte terminal differentiation, could be completely inhibited, this differentiation programme might be disrupted, and the malignant phenotype might then reappear in the non-malignant hybrids. This question was investigated further in the present paper. Total, and specific, inhibition of involucrin synthesis was indeed achieved by a sequential two-step antisense procedure, which might provide a systematic general method for the complete inactivation of other selected target genes.
Aortic endothelial cell heterogeneity in vitro Lack of association between morphological phenotype and collagen biosynthesis
Authors: Canfield A. E. , Wren F. E. , Schor S. L. , Grant M. E. , Schor A. M. 
Published: April 2021

ABSTRACT
Previous reports dealing with the characterisation of endothelial cells derived from the same tissue have produced apparently conflicting results in fundamental cellular attributes such as matrix biosynthesis and the ability to form sprouts in vitro. One potential explanation for this discrepancy is that endothelial cells actually comprise a heterogeneous population of cells displaying a significant degree of intra-site variation in phenotype. In order to address this question, we have characterised both cloned and uncloned lines of bovine aortic endothelial cells with respect to (a) their ability to adopt both the cobblestone and sprouting cell phenotypes and (b) matrix biosynthesis by cells displaying these two phenotypes. Data are presented indicating that all of the 18 cloned and 20 uncloned cell lines examined were capable of undergoing a reversible transition between the cobblestone and sprouting cell phenotypes in response to culture conditions. In all cases, sprouting occurred spontaneously in the presence of either serum or platelet-poor plasma and did not require the addition of exogenous factors to the medium. Twelve lines of cells were examined with respect to protein biosynthesis; these lines produced different types of collagens in differing proportions. The pattern of collagen synthesis displayed by every cell line was stable and did not vary with either passage number or batch of serum. The presence of a 3-D gel of native type I collagen increased specifically the synthesis of type IV collagen by one cell line. However, in four other cell lines, even though total synthesis was increased, the type of proteins secreted by these cells was not altered. Transition from a cobblestone to a sprouting cell phenotype by all five cell lines examined was not accompanied by any changes in the specific types of collagens synthesised by these cells. A decrease in thrombospondin and an increase in type IV collagen secretion was observed in one out of five cell lines. These data are discussed in terms of the possible significance of endothelial cell heterogeneity to the angiogenic response.
Cytostellin: A novel, highly conserved protein that undergoes continuous redistribution during the cell cycle
Authors: Warren Stephen L. , Landolfi Amalia S. , Curtis Carmel , Morrow Jon S. 
Published: April 2021

ABSTRACT
Cytostellin, a 240 kDa protein, has been purified from mammalian cells by immunoaffinity chromatography using monoclonal antibody H5. Immunofluorescence microscopy shows diffuse and punctate cytostellin immunoreactivity in interphase nuclei. Nuclease digestion and salt extraction are not required to expose the epitope. The onset of prophase is marked by the appearance of multiple intensely immunofluorescent cytostellin-containing ‘bodies’ within the nucleus. Nuclear disassembly is heralded by the movement of cytostellin bodies from the nucleus to multiple positions throughout the cell. Cytostellin bodies in metaphase, anaphase and telophase cells are widely dispersed, including some in cell processes far removed from the mitotic spindle apparatus. However, a distinct subset of larger, more intensely staining bodies surrounds the mitotic spindle apparatus. Cytostellin bodies remain in the cytoplasm of the daughter cells and disappear after the appearance of nascent nuclei. Cytostellin is immunologically distinct from other nuclear and cytoplasmic proteins, and it has been detected by immunoblot analysis in all species tested from yeast to humans. Based upon these findings, we postulate that cytostellin has a cell cycle-dependent function which is conserved in higher and lower eukaryotic cells.
Purification and immunological detection of pea nuclear intermediate filaments: Evidence for plant nuclear lamins
Authors: McNULTY A. K. , Saunders M. J. 
Published: April 2021

ABSTRACT
A major structural component of the inner face of the nuclear envelope in vertebrates and invertebrates is the nuclear lamina, an array of 1-3 extrinsic membrane proteins, lamins A, B and C. These proteins are highly homologous to intermediate filaments and are classified as type V. We report the first purification, antigenic characterization and immunocytochemical localization of putative plant lamin proteins from pea nuclei. We conclude that plant cells contain this ancestral class of intermediate filaments in their nuclei and that regulation of nuclear envelope assembly/disassembly and mitosis in plants may be similar to that in animal cells.
Indiscriminate synapsis in achiasmate Allium fistulosum L. (Liliaceae)
Authors: Jenkins G. , Okumus A. 
Published: April 2021

ABSTRACT
Seedlings of Allium fistulosum (2n=2x=16) were treated with aqueous colchicine with the intention of inducing tetraploidy. One treated, but undoubled, diploid mutant is described which consistently fails to form any chiasmata at diakinesis and metaphase I of meiosis. Electron microscopy of whole-mount surface-spread synaptonemal complex complements of pollen mother cell nuclei revealed that the achiasmate condition is probably due not only to the failure to complete synapsis, but also to the indiscriminate way in which the chromosomes form synaptonemal complexes during meiotic prophase. Synapsis begins and progresses with complete disregard to homology, with frequent exchanges of pairing partners resulting in the formation of multiple associations comprising heterologous chromosomes. Intrachromosomal synapsis is also evident as fold-back loops. Up to 78% of lateral element length is incorporated into synaptonemal complex, the morphology of which is not unlike that of normal A. fistulosum and other Allium species described previously. However, all the synaptonemal complexes are ineffective in terms of supporting chiasmata, since 16 univalents enter metaphase I and disjoin irregularly at anaphase I. The mutant is as a consequence completely male sterile. The synaptic behaviour observed confirms that the recognition of homology is an independent process and not a prerequisite for synaptonemal complex formation. It is hoped this mutant will be a valuable tool for probing the molecular basis of homology.
Attachment of human vascular smooth muscle cells to intact microfibrillar assemblies of collagen VI and fibrillin
Authors: Kielty Cay M. , Whittaker Stephen P. , Grant Michael E. , Shuttleworth C. Adrian 
Published: April 2021

ABSTRACT
Human vascular smooth muscle cells have been used to assess the implied role of connective tissue microfibrils as cellular ligands. Preparations of intact high-Mr microfibrillar assemblies of collagen VI and of fibrillin, respectively, were isolated from foetal bovine skin and used as ligands in cell attachment and spreading assays. Intact collagen VI microfibrils were capable of mediating cell attachment and partial spreading. Cell attachment assays using ligands composed of defined collagen VI fragments generated by pepsin or bacterial collagenase digestions demonstrated that both the triplehelical and non-collagenous domains of collagen VI had cell adhesion activity, although at reduced levels relative to intact microfibrils. Fibronectin was identified as a modulator of intact collagen VI microfibril-mediated cell attachment. These observations are indicative of complex multiple interactions between collagen VI microfibrils and smooth muscle cells. Purified fibrillincontaining microfibrils were also shown to support smooth muscle cell adhesion. Both pepsin-resistant and pepsin-sensitive domains of fibrillin exhibited some cell attachment activity, but at reduced levels relative to the intact fibrillin microfibrils. These data provide the first direct evidence of a physiological role for intact microfibrillar assemblies in cell-matrix interactions, and the involvement of integrin cell surface receptors containing the 1 subunit.
Total internal reflection fluorescence microscopy (TIRFM): II. *Topographical mapping of relative cell/substratum separation distances
Authors: Truskey G.A. , Burmeister J.S. , Grapa E. , Reichert W.M. 
Published: April 2021

ABSTRACT
A simplified model of TIRF optics was used to quantitate the relative membrane/substratum separation distances from the spatial pattern of TIRF image brightness. Phase-contrast and total internal reflection fluorescence microscopy (TIRFM) images were collected of bovine aortic endothelial cells (BAEC) plated onto glass microscope slides for 15 min, 30 min and 24 h. BAEC adherent for 15 min showed an absence of a focal contact morphology, with the region of closest apposition beneath the cell center. After 30 min, multiple contacts with the surface were established and the morphology became more irregular. BAEC attached for 24 h showed well-defined focal contact regions aligned in characteristically striated patterns. The relative distance between closest and farthest membrane/substratum separations are consistent with reported distance between focal and matrix contacts. Topographical maps of membrane/substratum separation distances over the entire ventral surface of the plated cells were constructed to demonstrate the utility of quantitative TIRF microscopy.
Comparative analysis of the effects of dimethyl sulfoxide and retinoic acid on the antigenic pattern of human ovarian adenocarcinoma cells
Authors: Grunt TH.W. , Somay C. , Oeller H. , Dittrich E. , Dittrich CH. 
Published: April 2021

ABSTRACT
HOC-7 malignant ovarian surface epithelial cells have been exposed to differentiation promoters like dimethyl sulfoxide (DMSO) and retinoic acid (RA) and the resulting cell phenotypes were characterized immunologically. Immunocytochemistry revealed that DMSO caused elevation of membrane-associated staining for epidermal growth factor-receptor (EGF-R) and for desmoplakins I and II (DPI+II). DMSO also stimulated cytoplasmic and surface labelling for CA 125 and extracellular deposition of fibronectin (FN). A fixed-cell ELISA system was used for quantification of these differentiation-like responses and revealed that DMSO efficiently induced expression of EGF-R, CA 125, FN and DPI+II in dosedependent manner. Immunocytochemistry, ELISA and Western blotting additionally demonstrated that both DMSO and RA caused down-regulation of myc oncoproteins. Densitometer evaluation of electrophoresed proteins revealed a 50% DMSO- and a 25% RA-induced myc reduction. Apart from growth reduction, which was seen for both inducers, inhibition of myc gene expression was the only response of HOC-7 cells to RA-treatment. The extent of myc down-regulation seems, therefore, to be crucial for the initiation of maturational processes in the cells. Subsequent phenotypic differentiation of HOC-7 cells causes elevated levels of EGF-R, CA 125, FN and DPI+II. This cell model might be useful for the distinction between induced growth reduction and differentiation of ovarian cancer cells.