bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interaction between vascular endothelial cells and vascular intimal spindle-shaped cells in vitro
Authors: Bono David De , Green Catherine 
Published: April 2021

ABSTRACT
The interactions between human or bovine vascular endothelial cells and fibroblast-like vascular intimal spindle-shaped cells have been studied in vitro, using species-specific antibodies to identify the different components in mixed cultures. Pure cultures of endothelial cells grow as uniform, nonoverlapping monolayers, but this growth pattern is lost after the addition of spindle cells, probably because the extracellular matrix secreted by the latter causes the endothelial cells to modify the way they are attached to the substrate. The result is a network of tubular aggregates of endothelial cells in a three-dimensional ‘polylayer’ of spindle-shaped cells. On the other hand, endothelial cells added to growth-inhibited cultures of spindle-shaped cells will grow in sheets over the surface of the culture. Human endothelial cells grown in contact with spindle-shaped cells have a reduced requirement for a brain-derived endothelial growth factor. The interactions of endothelial cells and other connective tissue cells in vitro may be relevant to the mechanisms of endothelial growth and blood vessel formation in vivo, and emphasize the potential importance of extracellular matrix in controlling endothelial cell behaviour.
Effects of ethidium bromide on cells of the cockroach central nervous system
Authors: Leech C. A. 
Published: April 2021

ABSTRACT
The effects of ethidium bromide were tested on isolated cockroach interganglionic connectives, either with the perineurium intact or desheathed. Intact connectives showed small, reversible depolarizations in response to 25 mM-ethidium. Desheathed connectives showed much larger, concentration-dependent depolarizations and the membrane potentials repolarized only slowly after washing out ethidium. The ethidium-induced depolarization could be blocked by raising the K concentration in the salines and was also shown to be reduced by rubidium, caesium, ammonium and ouabain. These treatments also reduced the degree of staining of nerve cords by ethidium, which was demonstrated to enter the cells.
Reduction in the mitochondrial membrane potential of Toxoplasma gondii after invasion of host cells
Authors: Tanabe Kazuyuki , Murakami Kenji 
Published: April 2021

ABSTRACT
The membrane potential of Toxoplasma gondii, an obligatory intracellular protozoan parasite, was monitored with the cationic permeant fluorescent dye rhodamine 123 (R123). Fluorescence microscopy revealed R123 to be partitioned predominantly in a restricted part of the parasite, which consisted of twisted or branched tubules, or of granular bodies. These structures were frequently connected to each other. The dye retention by these structures was markedly reduced by treating R123-labelled parasites with the proton ionophore, carbonylcyanide m-chlorophenylhydrazone, the potassium ionophore, valinomycin and the inhibitor of electron transport, antimycin A. Thus, these structures are regarded as the parasite mitochondria. Another cationic fluorescent dye, rhodamine 6G, stained the parasite mitochondria, whereas a negatively charged fluorescent dye, fluorescein, and the neutral compounds, rhodamine 110 and rhodamine B, did not. This fact indicates that R123 monitored the parasite mitochondrial membrane potential. T. gondii-infected 3T3 cells were also stained with R123. In contrast to the mitochondria of extracellular parasites, those of intracellular parasites failed to take up the dye. The absence of fluorescence in intracellular parasites persisted until the infected host cells ruptured and liberated daughter parasites 1 day after infection. Parasites, liberated from the host cells, either spontaneously or artificially by passing the infected cells through a 27G needle, regained the ability to take up the dye. After direct microinjection of R123 into the vacuole in which the parasite grows and multiplies, the dye appeared in the host-cell mitochondria but not in the parasite’s mitochondria. Thus, we conclude that the mitochondrial membrane potential of T. gondii was reduced after invasion of host cells by the parasite.
Repetitive titin epitopes with a 42 nm spacing coincide in relative position with known a band striations also identified by major myosin-associated proteins an immunoelectron-microscoplcal study on myofibrils
Authors: Fürst Dieter O. , Nave Rüdiger , Osborn Mary , Weber Klaus 
Published: April 2021

ABSTRACT
A direct titin-thick filament interaction in certain regions of the A band is suggested by results using four new monoclonal antibodies specific for titin in immunoelectron microscopy. Antibodies T30, T31 and T32 identify quasi-repeats in the titin molecule characterized by a 42-43 nm repeat spacing. These stripes seem to coincide with striations established by others on negatively stained cryosections of the A band. Antibodies T30 and T32 recognize epitopes matching five or two of the seven striations per half sacromere known to harbor both the myosin-associated C-protein and an 86K (K = 103 Mr) protein. Antibody T31 labels two stripes in the P zone, which correspond to the two positions where decoration is seen with 86K protein, but not with C-protein. The single titin epitope defined by antibody T33 is located 55 nm prior to the center of the M band. This position seems to coincide with the M7 striation defined by others on negatively stained A bands.
The T33 epitope position proves that the titin molecule, which is known to be anchored at the Z line, also penetrates into the complex architecture of the M band. The titin epitopes described here enable us to begin to correlate known ultrastructural aspects of the interior part of the A band with the disposition of the titin molecule in the sarcomere. They raise the question of whether there is a regular interaction pattern between titin and the thick filaments. Because of the limit of resolution of immunoelectron microscopy, it remains to be seen whether the repetitive titin epitopes of the C and P zones coincide exactly with the position of the myosin-associated proteins or lie on a similar lattice, out of register with these proteins.
Autoradiographic analysis of formylpeptide chemoattractant binding, uptake and intracellular processing by neutrophils
Authors: Janeczek Amelia H. , Marasco Wayne A. , Alten Pierson J. Van , Walter Robert J. 
Published: April 2021

ABSTRACT
Formylpeptide chemoattractant binds specifically to leukocyte cell surface receptors and during chemotactic activation, is internalized and processed by these cells. Using electron microscope autoradiographic techniques, we have examined the binding, uptake and disposition of fNle-Leu-Phe-Nle-[125I]Tyr-Lys by rabbit peritoneal neutrophils. Cells were incubated with ligand for 15 min at 4°C, rinsed and then further incubated in buffer at 4°C, 15°C, 24°C, or 37°C for 0–40min. For all cells incubated at 4°C, grains were predominantly localized on the plasma membrane. Initially, this formylpeptide was seen in small clusters or microaggregates that were not associated with coated pits. Upon further incubation at 37°C for 1 min, formylpeptide clustering on the plasma membrane increased and extensive internalization of formylpeptide was observed. Endocytosis of formylpeptide-receptor complexes clearly involved uncoated membrane pits and vesicles but did not appear to involve coated pits or coated vesicles. In the following 3 min, peptide proceeded in waves through compartments composed of small uncoated endocytic vesicles, then large vesicles, and then into dense granules. After 4 min at 37°C, the most active phase of intracellular processing subsided. The percentage of grains in the cytoplasm, granule and small vesicle compartments very gradually increased during the remainder of the 40 min incubation. Formylpeptide neither bound at 4 °C nor accumulated at 37 °C on the cell surface in proximity to the underlying Golgi/centrosome region of the cell. At 24°C, processing was slowed but formylpeptide-receptor complexes proceeded through a similar series of compartments. The q for formylpeptide uptake at 37 °C was 15-20 s, whereas at 24°C the q was approximately 10min. No uptake was observed at 15°C. The distinctive characteristics of formylpeptide binding and receptor-complex uptake seen here may be essential in initiating and maintaining continued chemotactic responsiveness.
The polarization of fibroblasts in early primary cultures is independent of microtubule integrity
Authors: Middleton C. A. , Brown A. F. , Brown R. M. , Karavanova I. D , Roberts D. J. H. , Vasiliev J. M. 
Published: April 2021

ABSTRACT
Fibroblasts in culture apparently require an intact system of microtubules in order to adopt and maintain a polarized morphology. In contrast, the polarization of a number of epithelial cell types has been shown to be microtubule-independent. Reconciliation of these apparently contradictory data is difficult, however, because the epithelial cells were studied in short-term primary cultures while the fibroblasts were studied in secondary or longer-term cultures. To clarify the situation we have examined the effects of the microtubule-disrupting drugs, colcemid and nocodazole, on the polarization of a single cell type, the chick heart fibroblast (HF), maintained in both primary (1°) and secondary (2°) cultures.
Immunofluorescence observations of both types of culture showed that in control medium the cells contained abundant microtubules, which were absent if the cells were cultured in medium containing either colcemid or nocodazole. The effects of microtubule-disrupting drugs on the polarization of the cells were quantified using two measures of cell shape, elongation and dispersion, both of which increase with increasing polarization. The results show that microtubule-disrupting drugs do not have a significant effect on the polarization of HF spreading in 1 ° culture but significantly reduce the polarization of HF spreading in 2° cultures. The effects of microtubule disruption on HF that had been maintained in 1° culture for 6h, 24 h or 48 h were also quantified. These measurements demonstrate that the polarization of HF in 1° culture becomes increasingly sensitive to microtubule disruption over this period until, after 48 h, they are as sensitive as HF in 2° cultures to microtubule disruption. The results show that the polarization of HF, cultured immediately after their isolation from the parent tissue is, like that of some epithelial cells, microtubule-independent but that during the first 48 h in culture these fibroblasts change in such a way that their polarization becomes microtubuledependent.
Ultrastructure and gold-immunolabelling of cell-substratum adhesions (podosomes) in rsv-transformed bhk cells
Authors: Gavazzi Isabella , Nermut Milan V. , Marchisio Pier Carlo 
Published: April 2021

ABSTRACT
Rous sarcoma virus-transformed BHK (RSV/B4-BHK) cells develop peculiar dot-like adhesions, that have been named podosomes, which, in the presence of serum, aggregate into ring- or crescentshaped adhesion sites, the rosettes of podosomes. We have used the lysis-squirting technique and gold-immunolabelling to study the 3D- organisation of podosomes and the location of vinculin, gelsolin, phosphotyrosine-containing proteins and pp60src at an ultrastructural level. Podosomes appear to be conical bodies, 0 ·l-0 ·5 μm high, made by a dense aggregation of actin oligomers and several associated proteins, connected in the rosette by actin filaments. Gelsolin and some phosphotyrosine-containing proteins are found within the podosomes, often associated with the actin filaments, while vinculin is found predominantly at the podosome periphery, associated with microfilaments, and pp60arc is located on the adjacent plasma membrane.
Three-dimensional localisation of DNA in the nucleolus of Spirogyra by correlated optical tomography and serial ultra-thin sectioning
Authors: Jordan E. Gwyn , Rawlins David J. 
Published: April 2021

ABSTRACT
Spirogyra nucleoli were shown by three-dimensional optical microscopy of DAPI fluorescence to contain DNA with a pattern and distribution matching those of the fibrillar centres. This was confirmed using different species with nucleoli showing different sizes of fibrillar centre. Much lower levels of fluorescence were seen corresponding to the dense fibrillar component. Nearly all the DAPI fluorescence arises from the fibrillar centres or from regions very close to their surface, indicating that this is the site of nucleolar transcription.
Appearance and origin of snRNP antigens in chick erythrocyte nuclei reactivated in heterokaryons
Authors: Bergman Mathias , Nyman Ulf , Ringertz Nils , Pettersson Ingvar 
Published: April 2021

ABSTRACT
Fusion of terminally differentiated chick erythrocytes (CE) with transcriptionally active rat myoblasts results in heterokaryons in which the CE nuclei undergo reactivation of RNA synthesis and splicing. In order to analyze the transport and assembly of small nuclear ribonucleoprotein (snRNP) particles and larger molecular complexes engaged in RNA processing, we have examined CE nuclei in heterokaryons for the presence of four U snRNP-related nuclear antigens (Sm, 70000Mr, F78 and M3G-cap) and for one antigen (La), associated with RNA polymerase III transcripts. Inactive erythrocyte nuclei showed low levels of Sm and F78 antigens, but the other antigens were undetectable. Immediately after fusion, the fluorescence of the pre-existing chicken Sm antigen was detected in the CEn, and then the intensity of the signal increased rapidly during reactivation. The other antigens appeared more slowly, reaching full intensity at different time points after fusion. Blocking of chick transcription did not block the appearance of Sm, 70000Mr, cap and La antigens but did effectively inhibit the appearance of the F78 antigen. It has previously been demonstrated that the structure recognized by this monoclonal antibody is physically associated with functional splicing complexes. Blocking of translation in heterokaryons abolished uptake of snRNPs into the chicken nuclei. Taken together, the results indicate that rat snRNP complexes were imported into the chick nuclei after assembly in the cytoplasm. For all the studied antigens, except F78, this translocation was independent of chick RNA synthesis. The appearance of the F78 antigen was totally dependent on expression of chicken genes.
A 116000 M
r nucleolar antigen specific for the dense fibrillar component of the nucleoli
Authors: Masson C. , Andre C. , Arnoult J. , Geraud G. , Hernandez-Verdun D. 
Published: April 2021

ABSTRACT
In ATT, a human autoimmune serum, we found anti-nucleolar antibodies that recognized nucleolar antigens confined to a single nucleolar compartment, the dense fibrillar component (DFC). We localized these antigens by immunoelectron microscopy in DFC of HeLa cell nucleoli both on Lowicryl sections and cryoultrathin sections without embedding. The antigens were solubilized by incubation with 2 M NaCl but not by RNase or DNase treatment. The ATT serum crossreacted with rat liver nucleoli and PtK1 cell nucleoli in which immunofluorescence labelling displayed a clumpy pattern. During mitosis, the antigens dispersed in the cytoplasm until late telophase, when they gathered in the prenucleolar bodies. In human peripheral lymphocytes, or HeLa cells treated with actinomycin D, the antigens were still present but the fluorescence intensity decreased. By immunoblotting using human nuclear extracts, the ATT serum bound to a 116 000 Mr protein at dilutions up to 1:2000. The reactivity of this band diminished with actinomycin D-treated nuclear extracts. Two minor bands were also observed at 97 and 70K (K=103Mr). Immunopurification by competition or elution demonstrated that the 116K antigens were at the origin of the nucleolar labelling. This DFC marker appeared to be different from the NORsilver-stained proteins, which in our preparations exhibited apparent molecular weights of 105,80 and 38 –40K. In addition, these 116K antigens did not exhibit the characteristics described for DNA topoisomerase I, fibrillarin or nucleolin. We propose the 116K antigen as a new marker of the DFC of the nucleoli.
Prompt heat-shock and heat-shifted proteins associated with the nuclear matrix-intermediate filament scaffold in Drosophila melanogaster cells
Authors: Ornelles David A. , Penman Sheldon 
Published: April 2021

ABSTRACT
Elevated temperatures induced the synthesis of several new proteins in Drosophila melanogaster cells. Besides the conventional heat shock (HS) proteins, another set of temperature-induced proteins has been found. These latter resemble the prompt HS proteins of mammalian cells. The prompt HS proteins of Drosophila differ from the well-known conventional HS proteins in the following properties: (1) synthesis of the prompt HS proteins is insensitive to the transcription inhibitor actinomycin D, which blocks the appearance of conventional HS proteins; (2) induction of the prompt HS proteins requires a significantly higher temperature than conventional HS proteins; (3) prompt HS proteins associate strictly with the nuclear matrix-intermediate filament complex (NM-IF), while the conventional HS proteins are found in all subcellular fractions; (4) prompt HS proteins of Drosophila are induced by high temperature alone while the conventional HS proteins are also produced by a variety of stress conditions.
Resinless-section electron micrographs show an altered nuclear matrix morphology in heat-shocked cells. The nuclear matrix fibers are altered in spatial distribution and have much additional electron-dense material. This added material probably reflects the soluble proteins shifted into the nuclear matrix at high temperature. The prompt HS proteins can be distinguished clearly from heat-shifted proteins by several criteria. Also, the prompt HS proteins are distinct from the heat-insensitive viral proteins of a persistent virus (HPS-1).
Nucleation of microtubules from mitotic centrosomes is modulated by a phosphorylated epitope
Authors: Centonze Victoria E. , Borisy Gary G. 
Published: April 2021

ABSTRACT
At the onset of mitosis a class of proteins appears that possess a phosphorylated epitope recognized by the monoclonal antibody MPM-2. Immunofluorescence staining shows that a subset of these proteins is associated with the centrosomes of the mitotic apparatus. The appearance of these proteins coincides with the increased microtubule nucleating capacity of the centrosomes. We have tested whether growth of microtubules from mitotic centrosomes in a lysed cell model is dependent on the availability of the phosphorylated epitope by blocking the epitope with a specific antibody or by modifying it by removal of the phosphate. Centrosomes incubated with purified tubulin nucleate microtubule asters. However, preincubating the centrosomes with MPM-2 blocks all microtubule nucleation. Pretreating mitotic centrosomes with alkaline phosphatase also inhibits nucleation. These data suggest that the phosphorylated epitope recognized by MPM-2 is important for microtubule nucleation.
Substratum-induced stress fiber assembly in vascular endothelial cells during spreading in vitro
Authors: Yost Jeffrey C. , Herman Ira M. 
Published: April 2021

ABSTRACT
We tested whether aortic endothelial cell (EC)-synthesized substrata, which modulate smooth muscle cell proliferation and EC motility following injury, could influence EC actin cytoskeleton and spreading in vitro. A partial characterization of the substrata indicates that the substratum prepared by deoxycholic acid extraction (DOC-derived substratum) is enriched with fibronectin and type IV collagen. Substratum prepared by removal of the intact monolayer with 20 mM EGTA in PBS (EGTA-derived substratum) contains fibronectin and heparan sulfate proteoglycan, but no type IV collagen. Morphometric analyses were performed on fixed and cytoskeletal antibody treated EC in order to quantitate the extent of spreading and stress fiber (SF) assembly. Compared to plastic, the DOC-derived substratum, a collagenase-treated DOC-derived substratum (CT-DOC-derived substratum) and the EGTA-derived substratum promote EC spreading 2·3-, 2·9- and 1·7-fold, respectively. In addition, there are 4·2-, 4·1- and 2·0-fold more SF on DOC-, CT-DOC- and EGTA-derived substrata, respectively, when compared to plastic. Subcellular fractionation and immunoprecipitation of cytoskeletal proteins from metabolically labeled EC were performed prior to electrophoresis and fluorography. The DOC-derived substratum increases immunoprecipitable actin and myosin 3-to 4.5-fold in both fractions compared to the EGTA-derived substratum and plastic. Collagenase treatment of the DOC-derived substratum partially inhibits this increase. Cycloheximide treatment prevents the rise in soluble actin and myosin as well as causing a reduction in SF number by 1/2 on the DOC-derived substratum and 2/3 on CT-DOC-derived substratum. We propose that fibronectin-collagen interactions are, in part, responsible for inducing endothelial synthesis of cytoskeletal proteins required for SF assembly. This substratum-induced actin-cytoskeletal reorganization facilitates EC spreading in vitro.
Ultrastructure of chromatin II. Three-dimensional reconstruction of Isolated fibers
Authors: Woodcock Christopher L. , McEwen Bruce F. , Frank Joachim 
Published: April 2021

ABSTRACT
Electron-microscope tomography has been used to reconstruct isolated, negatively stained chromatin fibers from Necturus maculosus erythrocytes. Tilt series micrographs from +70° to —70° at 5° intervals were obtained, allowing a reconstruction resolution of 3.3 nm for fibers lying parallel to the tilt axis. The fibers were found to be flattened in the plane of the carbon support, and also stained differentially according to the distance from the carbon. A number of methods of presenting the three-dimensional information were explored. Especially useful was an automatic peak search method for locating putative nucleosome positions coupled with the production of a computer-generated model. Other valuable techniques included the generation of projection stereograms and construction of solid models. A peripheral location of nucleosomes in the chromatin fiber was indicated, and helical arrangements of nucleosomes were observed over short regions. However, no long-range ordering of nucleosomes was apparent. The extent to which this lack of order may be the result of events occurring during the preparation of chromatin for electron microscopy is discussed.
Monoclonal antibodies to Leishmania mexicana promastigote antigens I. Secreted acid phosphatase and other proteins share epitopes with lipophosphoglycan
Authors: Ilg Thomas , Menz Beatrice , Winter Gerhard , Russell David G. , Etges Robert , Schell Dietmar , Overath Peter 
Published: April 2021

ABSTRACT
The abundant surface glycolipid, lipophosphoglycan (LPG), of Leishmania promastigotes is composed of phosphosaccharide repeating units linked via a phosphosaccharide core to a conserved lyso alkyl-phosphatidylinositol membrane anchor. It is shown in this paper that monoclonal antibodies (mAbs) directed against LPG also react with an acid phosphatase secreted by L. mexicana promastigotes. Acid phosphatase purified by column chromatography (apparent Mr=100000) reacts in immunoblots with the anti-LPG mAb AP3 and another mAb, L3.13, which does not recognize LPG. mAb L3.13 was used to purify the enzyme by affinity chromatography. The resulting glycoprotein has the same molecular weight and binds AP3 on immunoblots. The secreted phosphatase is non-covalently associated with a high molecular weight, galactose-containing glycan or proteoglycan that is recognized by both AP3 and L3.13. In addition to acid phosphatase, other parasite proteins appear to be modified by LPG epitopes.