bluuebunny/crossref_metadata_2025_split huggingface dataset:
Increased gap junction assembly between cultured cells upon cholesterol supplementation
Authors: Meyer Rita , Malewicz Barbara , Baumann Wolfgang J. , Johnson Ross G. 
Published: April 2021

ABSTRACT
Novikoff hepatoma cells provide an excellent model system for the study of gap junction assembly, a process that could be influenced by lipids and other factors at numerous points. Since it is possible to alter the cellular levels of cholesterol in these cells, it was added to the cells in serum-supplemented medium and changes in gap junction assembly were evaluated. Cells were dissociated and reaggregated following exposure to a range of cholesterol concentrations for 24 h. A five- to sixfold increase in the number of aggregated gap junction particles and a 50% increase in cellular cholesterol content were observed with 20 μM added cholesterol. A 1-h exposure to added cholesterol, during cell reaggregation, resulted in a fourfold increase in the number of aggregated gap junction particles, demonstrating that the effect was rapid. The number of aggregated gap junction particles and formation plaque areas were used as measures of junction assembly and assayed by quantitative freeze-fracture and electron microscopy. Junctional permeabilities were evaluated by means of dye transfer times following the intracellular microinjection of Lucifer Yellow. Increased dye transfer was observed between cholesterol-treated cells, which suggested that the increase in assembly was accompanied by an increase in junction permeability. Cells were treated with cycloheximide (100μgml−1) and actinomycin D (10 μgml−1) to determine whether protein and RNA syntheses were involved in the enhanced gap junction assembly. Cycloheximide but not actinomycin D blocked the increased junction assembly observed with added cholesterol. These results suggested that protein synthesis, but not RNA synthesis, is necessary for the increased gap junction formation observed. Thus, cholesterol added in the presence of serum-supplemented medium resulted in a dramatic and rapid increase in the assembly of functional gap junctions between Novikoff cells.
Growth factors for human fibroblasts in the solute remaining after clot formation
Authors: Gray Andrew J. , Reeves Jack T. , Harrison Nicholas K. , Winlove Peter , Laurent Geoffrey J. 
Published: April 2021

ABSTRACT
Fibroblasts adhere to, and readily grow into, fibrin clots that form as a result of the cleavage of fibrinogen by thrombin. Subsequent fibroblast replication is believed to be stimulated by mitogens released by entrapped platelets, such as platelet-derived growth factor. We suggest that the supernatant remaining after the fibrinogen-thrombin reaction could stimulate fibroblast replication, even in the absence of other blood components. To examine this hypothesis we expressed liquid from a fibrin clot and measured its mitogenic activity on human lung fibroblasts, in serum-free conditions, using a colormetric assay based on uptake and subsequent release of Methylene Blue. The clot supernatant caused a mitogenic response of 51±6 % above control and was equivalent to about half that elicited by medium containing 10 % newborn calf serum. On their own, both thrombin and fibrinopeptides A and B (small molecular weight cleavage products released from fibrinogen) showed some mitogenic activity, but there was also activity in higher molecular weight cleavage products, suggesting the presence of uncharacterised mitogens. It is proposed that these agents may play important roles in wound healing and diseases associated with vascular leakage and fibrosis, by stimulating fibroblast replication.
Spectrin, actin and the structure of the cortical lattice in mammalian cochlear outer hair cells
Authors: Holley M. C. , Ashmore J. F. 
Published: April 2021

ABSTRACT
Mammalian cochlear outer hair cells generate high-frequency forces in response to electrical stimulation. Force generation occurs in the lateral cortex of the cell, which includes the plasma membrane, a two-dimensional ‘cortical lattice’ of filamentous protein, and a multi-layered membrane system, the lateral cistemae. The cortical lattice is composed of relatively long filaments, 6.7 nm in diameter, which are wound circumferentially about the cell. These filaments are spaced about 42 nm apart and are cross-linked by a second type of filament 3.2 nm in diameter approximately aligned with the longitudinal axis of the cell. The cortical lattice is the only cortical structure that remains after the cell is fully extracted in the detergent Triton X-100 and high-salt solution. It retains the original cylindrical shape of the cell and is reversibly deformable. Antibodies raised against chicken gizzard actin, human blood spectrin and pig brain spectrin all react positively with the extracted lattice viewed using immunofluorescence. Three protein subunits identified in the organ of Corti have approximate molecular weights of 220, 235 and 240K (K= 103Mr) and react with the spectrin antibodies. A structural model of the lattice is proposed in which the circumferential filaments are composed of actin and the cross-links of spectrin. The model can account for the unusual cylindrical shape of outer hair cells and suggests a mechanism of force generation based upon the elastic and electrostatic properties of spectrin.
Import of proteins into chloroplasts
Authors: Flügge Ulf-Ingo 
Published: April 2021

ABSTRACT
Chloroplasts are the sites of photosynthesis in eukaryotic plants and are able to harvest solar energy for the synthesis of carbon skeletons. They, like mitochondria, are enclosed by two membranes; the outer and the inner envelope. The latter is the actual permeability barrier between the cytosol and the chloroplast stroma and the site of different metabolite translocators that coordinate the metabolism between compartments (Heldt and Flügge, 1987). Distinct from the envelope membranes are the energy-transducing thylakoid membranes that are located within the chloroplasts, and which enclose the thylakoid lumen. Stroma and thylakoid proteins each account for about 50% of the total chloroplast protein, whereas the two envelope membranes represent less than 1%. In spite of the chloroplasts’ semi-autonomy the majority of the chloroplasts proteins and also those of other cell compartments are coded for in the nucleus. These nuclear-coded proteins are synthesized in the cytosol, normally with N-terminal extensions called transit peptides, and are subsequently transported into the chloroplasts where they are processed to their mature sizes by specific proteases. Mechanisms must therefore exist to ensure that these proteins are (1) correctly targeted to chloroplasts and (2) subsequently localized into the correct chloroplast compartment: namely, the outer or inner envelope membrane, the intermembrane space, the chloroplast stroma, the thylakoid membrane or the thylakoid lumen. Major aspects of protein transport have been reviewed quite recently (Schmidt and Mishkind, 1986; Lubben et al. 1988; Keegstra et al. 1989). This Commentary will focus on the basic phenomena of this topic and will also present recent findings that extend the view of the reviews cited above.
The monovalent ionophore monensin maintains the nuclear localization of the human stress protein hsp28 during heat shock recovery
Authors: Arrigo AndrÉ-Patrick 
Published: April 2021

ABSTRACT
In HeLa cells exposed to supra-optimal temperatures, the a-crystallin-related stress protein hsp28 is reversibly redistributed inside the nucleus and increases its level of phosphorylation and aggregation. Here, I show that, at normal temperature after a heat stress, the sodium ionophore monensin maintains the nuclear localization of hsp28 without impairing the dephosphorylation of this protein. This phenomenon is not due to a prolongation, by monensin, of the synthesis of the heat-shock proteins after the heat stress. In contrast, the potassium ionophore nonactin induces only a weak alteration in the hsp28 locale, while the calcium ionophore A23187 and the uncoupler of oxidative phosphorylation FCCP have no effect. Following the removal of monensin 15 h after the heat stress, a further incubation of the cells for at least 36 h is necessary in order to observe a redistribution of hsp28 into the cytoplasm. A large fraction of hsp28 is then observed as dense excretion granules. In control cells kept at normal temperature, monensin, like nonactin, A23187 and FCCP, does not induce the redistribution of hsp28 inside the nucleus. Taken together, these results suggest that the disruption of the Na+ active transport by monensin probably inhibits the redistribution of hsp28 in the cytoplasm after heat shock.
Ribonuclease H(70) is a component of the yeast nuclear scaffold
Authors: Karwan Robert M. , Laroche Thierry , Wintersberger Ulrike , Gasser Susan M. , Binder Maximilian 
Published: April 2021

ABSTRACT
We have used monospecific antibodies against three ribonuclease H enzymes of Saecharomyces cerevisiae to investigate their intracellular localization. Fractionation experiments, as well as immunocytochemical staining, revealed a predominantly cytoplasmic localization of the RNase H proteins of 42 000 and 70 000, Mr, whereas that of 55000 Mr showed equal distribution between nuclei and cytoplasm. The nuclear moiety of ribonuclease H(70) was found to be a part of the yeast nuclear scaffold, as investigated by immunoblotting and antibody inhibition experiments. The 42000 and 55000 Mr enzymes, on the other hand, are not scaffold-associated. We conclude that RNase H(70) is part of the nuclear substructure of yeast that was previously found to maintain specific interactions with yeast chromosomal origins of replication (ARS elements).
Mutations at the ASP locus of Drosophila lead to multiple free centrosomes in syncytial embryos, but restrict centrosome duplication in larval neuroblasts
Authors: Gonzalez C. , Saunders R. D. C. , Casal J. , Molina I. , Carmena M. , Ripoll P. , Glover D. M. 
Published: April 2021

ABSTRACT
Mutations at abnormal spindle result in abnormally long and wavy microtubules in the meiotic spindles of males. Some of these spindles have a single pole and take the form of unopposed hemi-spindles. Unfertilised eggs produced by homozygous asp females may have either no nuclei, or a small number of large nuclei, consistent with there also being an effect upon female meiosis. Such eggs also display free centrosomes and independent arrays of microtubules. Embryos that have this phenotype are also present among the progeny of fertilised homozygous asp females, together with embryos that undergo varying degrees of aberrant morphogenesis, developing a variety of abnormal cuticle patterns. This latter category shows asynchronous mitoses prior to cellularisation, and has abnormal arrays of spindle microtubules. Such embryos can develop large areas that are either devoid of or have a reduced number of nuclei, in which there are centrosomes that have dissociated from the mitotic spindles. Neuroblasts in the brains of homozygous asp larvae display a high mitotic index, and have condensed chromosomes aligned as if blocked at metaphase. Immunostaining reveals that many cells contain a single centrosome connected to the metaphase chromosomes by microtubules in a hemi-spindle-like structure.
Neurofilament proteins are co-expressed with desmin in heart conduction system myocytes
Authors: Vitadello Maurizio , Matteoli Michela , Gorza Luisa 
Published: April 2021

ABSTRACT
We have recently shown that specialized myocytes of the rabbit heart express a cytoskeletal protein similar to the M subunit of neurofilaments (NF). Since this result was obtained using a single anti-NF-M monoclonal antibody, we tested on conduction myocytes a panel of five anti-NF antibodies, specific for each of the three NF subunits and for phosphorylated and non-phosphorylated epitopes. Two antibodies, one specific for the L subunit and one for phosphorylated M subunit of NF, reacted with specialized myocytes in immunohistochemistry. In immunoblots on conduction tissue homogenates the two antibodies recognized two polypeptides with electrophoretic mobility and solubility properties identical to those of NF-L and NF-M in the sciatic nerve.
The subcellular distribution of NF immunoreactivity in specialized myocytes was very similar to desmin localization; namely, it was distributed on large filamentous bundles and on fine filaments localized transversely at the level of the Z line. At the ultrastructural level, immunoreactive filaments were localized in the intermyofibrillar space and connected myofibrils with mitochondria. Co-expression of NF proteins and desmin was also observed in vitro in a minor population of cardiac myocytes cultured from embryonic rabbit heart. In most cases NF immunoreactivity co-localized with desmin, especially where filaments were well organized, but in some cells anti-NF and anti-desmin antibodies labelled different filamentous structures.
These results indicate that NF proteins are structural components of the cytoskeleton of specialized myocytes and show a subcellular distribution very similar to desmin. Such a composition of intermediate filaments indicates that in these cardiac cells muscle differentiation is compatible with the expression of neuronal proteins.
Adsorptive endocytosis and membrane recycling by cultured primary bovine brain microvessel endothelial cell monolayers
Authors: Raub Thomas J. , Audus Kenneth L. 
Published: April 2021

ABSTRACT
The dynamics of membrane recycling were examined in primary cultures of brain microvessel endothelial cells (BMECs). Because the BMEC surface was dominated by galactosylated glycoconjugates, ricin agglutinin (RCAI) was used as a tracer to follow the endocytosis and recycling of RCAI binding sites. These binding sites accounted for 75% of the iodinatable or most externally disposed plasma membrane proteins. Because greater than 90% of the RCAI that had bound to BMECs was removed by a brief, nontoxic treatment with galactose, the amounts and kinetics for internalization and efflux of [125I]RCAI were measured. Both endocytosis and efflux were energy dependent. By using pseudo-first-order kinetics, the t 1 values for RCAI binding, internalization and efflux were 5, 18 and 13–14 min, respectively. By comparing efflux with and without galactose present, we found that 60% of the RCAI binding sites that had been internalized were returned to the cell surface and reinternalized. Quantifying the distribution of gold-RCAI following internalization showed kinetics consistent with that obtained using radiolabeled RCAI. Both horseradish peroxidase (HRP) and gold-conjugated RCAI that had bound BMEC at 4 °C became localized within more caveolae within 2.5min of warming to 37°C to permit endocytosis. With time, RCAI appeared within endosomes and tubules and vesicles of which some were located in the trans-Golgi network (TGN). The distribution of HRP-RC Al contrasted with that of free HRP, which was not routed to the TGN. The absence of RCAI conjugates in association with the basolateral membrane domain suggested the presence of functional tight junctions and maintenance of polarity throughout the duration of these experiments. These results showed that membrane recycling was more extensive and much slower than fluid-phase endocytosis in cultured BMECs. Moreover, we found that endocytosis of membrane by BMECs in culture was similar to that reported for brain endothelium in vivo in that a fraction of the cell surface membrane was routed to the TGN.
Laminin potentiates differentiation of PCC4uva embryonal carcinoma into neurons
Authors: Sweeney T. M. , Ogle Roy C. , Little C. D. 
Published: April 2021

ABSTRACT
The embryonal carcinoma PCC4uva differentiates into neurons in response to treatment with retinoic acid and dbcAMP. We used this in vitro model system to study the effects of laminin on early neural differentiation. Laminin substrata markedly potentiate neural differentiation of retinoic acid and dbcAMP-treated cultures. Only laminin induced more rapid neural cell body clustering, neurite growth and neurite fasciculation as compared to type IV collagen, type I collagen, and fibronectin substrata. Exogenous laminin substrata promoted greater cell attachment, cellular spreading and growth to confluence than type IV collagen, type I collagen, fibronectin and glass substrata. Laminin-induced effects were inhibited by addition of laminin antibodies or the synthetic laminin-derived peptide De-Gly-Ser-Arg-NH2 (YIGSR-NH2). Treatment with YIGSR-NH2also inhibited neural differentiation in the absence of exogenous laminin substrata, whereas synthetic peptides containing the RGD sequence and a control peptide YIGSK-NH2 showed no inhibitory effects. These results are consistent with the hypothesis that specific interactions between an early differentiating cell population(s) and extracellular laminin are required during neural differentiation.
Blood platelet formation in vitro. The role of the cytoskeleton in megakaryocyte fragmentation
Authors: Tablin Fern , Castro Marlene , Leven Robert M. 
Published: April 2021

ABSTRACT
We have developed a unique in vitro model that promotes differentiation of megakaryocytes into platelets. When megakaryocytes isolated from guinea pig bone marrow were cultured on hydrated rat tail collagen gels, cells spontaneously formed elongated, beaded processes that fragmented to yield cytoplasmic pieces with the same size and internal composition as individual platelets. Addition of nocodazole at the initiation of cultures blocked process formation, while addition of nocodazole to cells with previously established processes resulted in their retraction. The addition of taxol to cultures resulted in abnormally thick processes that were tightly adherent to the underlying substratum, and did not bead or fragment Cytochalasin D accelerated process formation and fragmentation of megakaryocytes cultured on collagen gels by twofold. On the basis of these results, we propose a model for platelet formation in culture that involves the following steps: adherence of megakaryocytes to the underlying extracellular matrix; dilation of the demarcation membrane system and breakdown of the actin-rich peripheral zone; microtubule-based extension of pseudopodia, which are no longer adherent to the substratum; and fragmentation into platelets by the coalescence and fusion of demarcation membrane vesicles with the plasma membrane. We feel that this distinctive culture system closely approximates thrombocytopoiesis in vivo, thus allowing detailed elucidation of this important process.
Localization of platelet-derived growth factor (PDGF) in CHO cells transfected with PDGF A- or B-chain cDNA: Retention of PDGF-BB in the endoplasmic reticulum and Golgi complex
Authors: Thyberg Johan , Östman Arne , Bäckström Gudrun , Westermark Bengt , Heldin Carl-Henrik 
Published: April 2021

ABSTRACT
Platelet-derived growth factor (PDGF) is a powerful mitogen for connective tissue cells. It is made up of two polypeptide chains (A and B) and exists in three dimeric forms (AA, AB, and BB). Transfection experiments have indicated that PDGF-AA and -AB are secreted as 30×103Mr products, whereas PDGF-BB is processed into a 24×103Afr product and remains associated with the cells. Here, CHO cells were transfected with PDGF B- or A-chain cDNA and the intracellular distributions of the respective gene products were compared by indirect immunofluorescence and immunoelectron microscopy, using primary antibodies specific for PDGF B- and A-chain homodimers. PDGF-BB was most conspicuous in stacked Golgi cisternae. It was also found in the endoplasmic reticulum and in lysosomes. Upon treatment of the cells with the microtubule-disruptive drug nocodazole, the Golgi complex was broken up and its stacks of cisternae were dispersed throughout the cytoplasm together with clusters of lysosomes. After this structural disorganization, the concentration of PDGF-BB to the Golgi stacks was even more prominent than before. Weak reactivity for PDGF-AA was detected in the endoplasmic reticulum and groups of vacuoles, both in control and nocodazole-treated cells, whereas Golgi stacks and lysosomes only seldom were positive. The observations suggest that PDGF-BB is processed and retained within the endoplasmic reticulum and Golgi complex. Eventually, it may also be transferred to lysosomes for degradation. In contrast, PDGF-AA is likely to follow a pathway for bulk flow, including rapid passage through the endoplasmic reticulum and Golgi complex, package in secretory vacuoles, and extracellular release by exocytosis.
Characterization of the ZO-1 protein in endothelial and other cell lines
Authors: Li Changxin , Poznansky Mark J. 
Published: April 2021

ABSTRACT
A high molecular weight tight junction-associated protein, ZO-1, has been demonstrated in liver (hepatocytes) and in both epithelium and endothelium. We carried out studies to examine the presence of the protein in vascular endothelial cell cultures and several other types of cultured cells, and the relationship between the ZO-1 protein content and confluency of endothelial cell monlayers. Immunofluorescence labelling of endothelial monolayers and two types of epithelial monolayers, IEC-6 and MDCK, with monoclonal antibody against ZO-1 protein localized the protein to the cell peripheries. Its association with the cell periphery only occurred when cells had contact with one another as demonstrated in endothelial cells. We have been able to show a positive correlation between the ZO-1 content of the cells and the extent of monolayer confluency in the endothelial cells by immunoblotting. The protein is much less expressed in nonconfluent endothelial cell monolayers and totally absent from mouse myeloma cultures. The presence and confluence-related expression of the protein in endothelium give support to the hypothesis that tight junctions exist in confluent endothelial cells and that the ZO-1 protein is expressed under the conditions where tight junction interactions occur.
Assessing the differentiation state of cultured bovine urothelial cells: elevated synthesis of stratification-related K5 and K6 keratins and persistent expression of uroplakin I
Authors: Surya Babu , Yu Jun , Manabe Motomu , Sun Tung-Tien 
Published: April 2021

ABSTRACT
Although significant progress has recently been made in culturing mammalian urothelial cells, relatively little is known about their biochemical differentiation. In this paper, we assessed the differentiation state of cultured bovine urothelial cells by analyzing their keratins and a cell surface marker, uroplakin I. Urothelial cells were serially cultured either in a serum-free medium, or in a serumcontaining medium in the presence of 3T3 feeder cells, with similar results. Despite their stratified appearance, both normal urothelium and cultured urothelial cells synthesize mainly K8, K18 and K19, keratins that are typically seen in simple epithelia. However, cultured urothelial cells synthesize a greatly increased amount of K5 and K6 keratins, which are usually expressed by stratified epithelia but present only in trace amounts in normal urothelium. These data indicate that, as far as keratin synthesis is concerned, cultured urothelial cells undergo an altered pattern of differentiation towards a more ‘stratified phenotype’; this unusual finding has interesting implications for urothelial evolution. In the meantime, many superficial cells in cultured urothelial colonies make uroplakin I, a 27 × 103Mr protein subunit of the asymmetrical unit membrane (AUM) characteristic of urothelial (superficial) umbrella cells. These results indicate that cultured urothelial cells undergo, at least in part, AUM biogenesis. Cultured urothelial cells thus provide a useful experimental model system for studying certain early steps of AUM formation.