bluuebunny/crossref_metadata_2025_split huggingface dataset:
Membrane-binding properties of filensin, a cytoskeletal protein of the lens fiber cells
Authors: Brunkener Mariann , Georgatos Spyros D. 
Published: April 2021

ABSTRACT
Filensin is a 100/110 kDa membrane-associated protein found in lens fiber cells. Previous studies have shown that this protein polymerizes in vitro and binds strongly to vimentin and to another 47 kDa lens membrane protein. Using cosedimentation assays, flotation assays and immunoelectron microscopy, we have examined the properties of purified filensin and measured its binding to lens membranes. Filensin behaves as a urea-extractable, hydrophilic protein which does not partition with Triton X-114 and is not affected by 1 M hydroxylamine at alkaline pH, an agent known to release fatty-acylated proteins from the membrane. Immunoblotting of urea-extracted lens membranes with two different affinity-purified antibodies reveals that, unlike intact filensin, a COOH-terminal filensin degradation product (51 kDa) remains tightly associated with the membranes. Purified filensin binds directly to urea-stripped lens membranes, but not to protein-free vesicles reconstituted from total lens lipids. The binding of filensin is not significantly influenced by the purified 47 kDa protein. Interestingly, the filensin-binding capacity of urea-extracted membranes is increased at least twofold after trypsin treatment, which removes entirely the 51 kDa peptide from the membranes and presumably unmasks additional filensin-acceptor sites. Consistent with this, filensin binds to trypsinized and non-trypsinized membranes with similar affinities (2×10−7 and 4×10−7 M, respectively). Treatment of the membranes with thrombin, which also eliminates the 51 kDa peptide, does not increase their binding capacity, apparently because filensin-acceptor sites are also destroyed during proteolysis. Finally, heat-treatment of the trypsinized membranes, or digestion of urea-stripped membranes with a-chymotrypsin and V8 protease, affect filensin binding to a variable degree. Based on these data, we conclude that filensin and its COOH-terminal proteolytic product have the potential to directly associate with intrinsic elements of the lens cell membrane.
Tenascin in bone morphogenesis: expression by osteoblasts and cell type-specific expression of splice variants
Authors: Mackie Eleanor J. , Tucker Richard P. 
Published: April 2021

ABSTRACT
The extracellular matrix glycoprotein, tenascin, is associated in vivo with mesenchyme undergoing osteogenesis and chondrogenesis, but is absent from mature bone and cartilage matrix. The expression of tenascin by osteoblastic cells in vitro has been investigated by immunoblotting and immunocytochemistry. Tenascin was secreted into the medium and deposited in the matrix by human and rat osteoblast-like cell lines, as well as by primary osteoblast-enriched cultures from chick embryo calvarial bones. In primary osteoblast-enriched cultures, extracellular tenascin was found only in cell aggregates expressing the osteoblast marker alkaline phosphatase. Chicken osteoblast cultures synthesized almost exclusively the largest tenascin subunit, whereas fibroblast cultures from periostea of chicken calvariae synthesized approximately equal amounts of all three subunits. In situ hybridization studies of developing chicken bones, using a cDNA probe that hybridizes to all chicken tenascin splice variants, showed specific labelling of both osteogenic and chondrogenic regions of developing endochondral bones. In contrast, a cDNA probe specific for the large tenascin splice variant showed specific hybridization in osteogenic but not chondrogenic regions. Within osteogenic regions, tenascin mRNA was expressed by osteoblasts. A comparison of in situ hybridization and immunohistochemical studies demonstrated that tenascin mRNA and protein were codistributed in osteogenic regions of endochondral and membrane bones, whereas protein was retained in regions of differentiating cartilage where mRNA was no longer detectable. The results presented here demonstrate that tenascin is synthesized by osteoblasts. Moreover, within developing bones, there are at least three different cell type-specific patterns of expression of tenascin splice variants.
Adhesion of golgi cisternae by proteinaceous interactions: intercisternal bridges as putative adhesive structures
Authors: Cluett Edward B. , Brown William J. 
Published: April 2021

ABSTRACT
We have investigated the nature of the component(s) responsible for holding the cisternal membranes of the Golgi complex into a stacked unit. Isolated Golgi complexes were treated with a variety of agents to induce the separation of intact Golgi stacks into single cisternal elements, i.e. “unstacking”, and the effects were analyzed and quantitated by electron microscopy. In control experiments, isolated, intact Golgi stacks were stable at 4°C and 20°C for ≥1 h; however, some unstacking occurred at 32°C. Treatment of intact Golgi stacks with a variety of proteolytic enzymes resulted in a time- and dose-dependent unstacking of the cisternae, although stacks were resistant to various other proteases. Following liberation from the stack, single cisternae remained flattened with dilated rims. The integrity of intact Golgi stacks was unaffected by treatment with various concentrations and combinations of monovalent and divalent cations, or chelators of divalent cations. Electron microscopic observations of tannic acid- or negatively stained Golgi complexes, revealed the presence of highly structured, intercisternal “bridges”. When seen within intact Golgi complexes, these bridges were only consistently found between closely apposed cisternae and were not observed on dilated rims or secretory vesicles. These bridges, on both intact stacks and physically disrupted cisternae, were rectangular, being ∼8.5 nm in width, ∼11 nm in height. Treatment with proteases under conditions that resulted in the unstacking of intact complexes also removed these bridge structures. These data show that proteinaceous components are responsible for holding Golgi cisternae together into a cohesive, stacked unit, and identify a candidate bridge structure that could serve this purpose.
Cell shape, chromosome orientation and the position of the plane of division in Vicia faba root cortex cells
Authors: Oud J. L. , Nanninga N. 
Published: April 2021

ABSTRACT
Three-dimensional chromosome orientation was studied in thick sections of Vicia faba root meristem, using confocal microscopy and digital image analysis techniques. In the proliferative part of the root meristem, where the cells are organized in longitudinal files, it was expected to find dividing cells with a spindle axis parallel to the file axis and, occasionally, perpendicular to the file axis (resulting in a local file bifurcation). However, we observed a large number of oblique spindle axes. From metaphase to telophase there was a progressive increase in the rotation of the spindle axis. A 90° turn of the metaphase equator plane was never observed. Threedimensional measurements of both the space occupied by the ana- and telophase chromosome configurations, and the size of the corresponding cortex cells, showed that most cells were too flat for an orientation of the spindle parallel to the file axis. Apparently, cell size limitations forced the spindle to rotate during mitosis. Consequently, the nuclei in the daughter cells were positioned diagonally in opposite directions, instead of on top of each other. In the majority of these cells, a transverse plane of division would intersect the nuclei. Therefore, the new cell wall was sigmoid shaped or oblique. Most daughter cells remained within the original cell file but, occasionally, in extremely flat cells the position of the daughter nuclei forced the cell to set a plane of division parallel to the file axis. This resulted in file bifurcation. It has been concluded that cell shape, the extent of spindle rotation and the position of the division plane are related.
Dynamics of three-dimensional replication patterns during the s-phase, analysed by double labelling of dna and confocal microscopy
Authors: Manders E. M. M. , Stap J. , Brakenhoff G. J. , Driel R. Van , Aten J. A. 
Published: April 2021

ABSTRACT
The temporal and spatial progression of DNA replication in interphase nuclei of eukaryotic cells has been investigated. Application of a recently developed technique for the immunofluorescence double staining of cell nuclei labelled first with iododeoxyuridine (IdUrd) and subsequently with chlorodeoxyuridine (CldUrd) allows the visualization of two replication patterns in the same nucleus originating from two different periods of the S-phase. We have analysed changes in the three-dimensional replication patterns during the S-phase. To record dual colour three-dimensional images of doubly stained nuclei, a confocal microscope is used. This CSLM is equipped with a specific laser/filter combination to collect both fluorescence signals (FITC and Texas Red) in a single scan, thus precluding pixel shift between the images. A method for the quantitative evaluation of the degree of overlap between DNA regions replicated in two different periods of the S-phase is applied. The results confirm the generally accepted theory that DNA is replicated coordinately in a specific temporal order during the S-phase. The replication time of a DNA domain (i.e. the time between initiation and termination of DNA replication within a domain) at the very beginning of the S-phase was known to be one hour (Nakamura et al., 1986). Our observations show that in the rest of the S-phase, the replication time of a DNA region is also about one hour. We conclude that replicon clusters located in the same region are replicated in the same relatively short period of time. After this period there is no unreplicated DNA left in this region.
DNA replication in chloroplasts
Authors: Heinhorst Sabine , Cannon Gordon C. 
Published: April 2021

ABSTRACT
Chloroplasts contain multiple copies of a DNA molecule (the plastome) that encodes many of the gene products required to perform photosynthesis. The plastome is replicated by nuclear-encoded proteins and its copy number seems to be highly regulated by the cell in a tissue-specific and developmental manner. Our understanding of the biochemical mechanism by which the plastome is replicated and the molecular basis for its regulation is limited. In this commentary we review our present understanding of chloroplast DNA replication and examine current efforts to elucidate its mechanism at a molecular level.
Thermotolerance in mammalian cells protein denaturation and aggregation, and stress proteins
Authors: Kampinga Harm H. 
Published: April 2021

ABSTRACT
Cells that have been pre-exposed to thermal stress can acquire a transient resistance against the killing effect of a subsequent thermal stress. The cause for this phenomenon, called thermotolerance, seems to be an enhanced resistance of proteins against thermal denaturation and aggregation. This resistance can be expressed as an attenuation of damage formation (less initial damage) or as a better repair of the protein damage (facilitated recovery). Heat Shock (or better, Stress) Proteins (HSPs) may play a role in and even be required for thermal resistance. However, rather than stress-induced enhanced synthesis and elevated total levels of HSPs per se, the concentration of, both constitutive and inducible, HSPs at and/or (re)distributed to specific subcellular sites may be the most important factor for the acquisition of thermotolerance. Specific HSPs may be involved either in damage protection or in damage repair.
Microtubule nucleating activity of centrosomes in cell-free extracts from Xenopus eggs: involvement of phosphorylation and accumulation of pericentriolar material
Authors: Ohta Kunihiro , Shiina Nobuyuki , Okumura Eiichi , Hisanaga Shin-Ichi , Kishimoto Takeo , Endo Sachiko , Gotoh Yukiko , Nishida Eisuke , Sakai Hikoichi 
Published: April 2021

ABSTRACT
We have studied the regulation of microtubule nucleating activity of the centrosome using cell-free extracts from Xenopus eggs. We found that the number of microtubules per centrosome increases dramatically with time during incubation of isolated centrosomes in interphasic egg extracts prepared 20-30 minutes after electric activation of cytostatic factor (CSF)-arrested eggs. The increase in microtubule nucleation was still conspicuous even when KCl-treated centrosomes (centrosomes stripped of their microtubule nucleating activity by 1 M KCl treatment) were incubated in interphasic extracts. Electron microscopy and immunostaining by anti--tubulin and 5051 human anti-centrosome antibodies revealed that pericentriolar material (PCM) was accumulated during the increase in microtubule nucleation from centrosomes in interphasic extracts, suggesting regulation of centrosomal activity by PCM accumulation. The ability of egg extracts to activate microtubule nucleation from centrosomes was also assumed to be regulated by phosphorylation, since addition of protein kinase inhibitors into interphasic extracts totally blocked the increase in microtubule nucleation from the KCl-treated centrosome. The ability of CSF-arrested mitotic extracts to increase microtubule nucleation from KCl-treated centrosomes was 3.5-to 5-fold higher than that of interphasic extracts, while PCM accumulation in mitotic extracts seemed to be similar to that in interphasic extracts. The increase in microtubule nucleation from KCl-treated centrosomes was strikingly enhanced by the addition of purified p34cdc2/cyclin B complex to interphasic extracts, but not by MAP kinase, which is activated downstream of p34cdc2/cyclinB. These results suggest two pathways activating centrosomal activity in egg extracts: accumulation of PCM and phosphorylation mediated by p34cdc2/cyclin B.
The gene encoding the calcium binding protein calcyclin is expressed at sites of exocytosis in the mouse
Authors: Timmons Paula M. , Chan C.-T. J. , Rigby Peter W. J. , Poirier Françoise 
Published: April 2021

ABSTRACT
Calcyclin is a member of the S100 family of calcium binding proteins. We have found by in situ hybridization that calcyclin transcripts are restricted to specific cell types within a limited number of mouse organs. High levels of expression in the epithelia lining the gastrointestinal, respiratory and urinary tracts, and specific localization of the transcripts to the goblet cells in the small intestine, lead us to suggest a role for calcyclin in the process of mucus secretion. In addition, calcyclin expression was detected in the corpus luteum, placenta and nerves within the gut wall, which are all sites of regulated exocytosis. We propose that this S100-like protein may be part of a calcium signalling pathway utilized in the secretion of various products by different cell types.
The membrane fusion events in degranulating guinea pig eosinophils
Authors: Lindau M. , Nüsse O. , Bennett J. , Cromwell O. 
Published: April 2021

ABSTRACT
We have investigated the granule fusion events associated with exocytosis in degranulating peritoneal guinea pig eosinophils by time-resolved patch-clamp capacitance measurements using the phase detector technique. Intracellular stimulation with micromolar calcium and GTPγS induces a 2-to 3-fold capacitance increase. The main phase of the capacitance increase occurs after a delay of 2-7 minutes and is composed of well-resolved capacitance steps. The number of steps is very close to the number of crystalloid granules contained in a resting cell and the step size distribution with a peak at 9 fF is in excellent agreement with the granule size distribution determined by electron microscopy. The individual granules thus fuse sequentially with the plasma membrane. The stepwise capacitance increase is frequently preceded by an apparently continuous capacitance increase which consists of steps smaller than 4 fF, indicating exocytosis of small vesicles as distinct from crystalloid-containing granules. In some cases the time course of the opening of individual fusion pores could be recorded, and this revealed metastable conductance states below 300 pS but random fluctuations at higher conductance levels. This behaviour suggests that the small fusion pore might be a protein structure similar to an ion channel, which becomes a continuously variable lipid pore at higher conductances.In some cells a significant capacitance decrease was observed which is apparently continuous, suggesting a process of membrane uptake by endocytosis of small vesicles.
Human retinal pigment epithelial cells from different donors continuously produce a vascular endothelial cell-stimulating factor into serum-free medium
Authors: Yang Q. R. , Smets R. M. E. , Neetens A. , Berghe D. Vanden 
Published: April 2021

ABSTRACT
Mitogenic activities of human retinal pigment epithelial cell-conditioned medium (HRPE-CM) with different effects, such as inhibition, stimulation or no effect, on the proliferation of vascular endothelial cells (EC) in vitro have been reported. In this study, 14 HRPE cell lines were established from normal human eyes. Human umbilical vein endothelial cells (HUVEC) in the early passages were used as target cells to detect the mitogenic activity of HRPE-CM on the growth of vascular EC. Our results confirm that HRPE cells in culture continuously synthesize and secrete HUVEC growth substance(s) into a serum-free medium. The ability of HRPE cell lines to produce this mitogen seem unrelated either to in vivo donor factors or to in vitro cell life span. Using an enzyme-linked immunosorbance assay, we demonstrated that only HRPE cell extract, not HRPECM, can be recognized by basic fibroblast growth factor (bFGF)-specific antibody, though identical bioactivities on the growth of HUVEC were found in both preparations. The active component in HRPE-CM was heat- and trypsin-sensitive, and stable at extremes of pH (2.5 to 10.0). In addition, the bioactive molecule could not pass through a Mr 30,000 cut-off membrane, suggesting that it is a fairly high molecular mass polypeptide. These observations suggest that the EC growth factor in HRPE-CM is distinct from fibroblast growth factors (FGFs).
Proliferative behaviour of high-ploidy cells in two murine tumour lines
Authors: Hoz Carmen de la , Baroja Antonio 
Published: April 2021

ABSTRACT
The presence of high-ploidy cells in malignant tumours has long been documented. However, the biological significance of these cells is not known and there is a great deal of controversy over their proliferative potential. We have analysed the behaviour of these cells in two murine tumour lines, B16F10 melanoma and 3T3A31M angiosarcoma, determining their DNA content by microspectrophotometry and using time-lapse film studies. We have found a discrepancy between the presence of high-ploidy cells in metaphase and the absence of hyperploid telophases. High-ploidy metaphases may be aborted (mitotic polyploidization), prolonged in time or evolve in the form of multipolar, generally tripolar, mitoses. Our results suggest that high-ploidy cells are capable of proliferating, despite certain peculiarities in their cell cycle, and constitute a tumour subpopulation whose role in neoplasia merits further study.
Immortalization of polarized rat retinal pigment epithelium
Authors: Nabi Ivan R. , Mathews Abraham P. , Cohen-Gould Leona , Gundersen Doris , Rodriguez-Boulan Enrique 
Published: April 2021

ABSTRACT
Rat retinal pigment epithelial (RPE) cells were immortalized by infection with a temperature-sensitive tsA SV40 virus and following cloning and selection for epithelial properties the polarized RPE-J cell line was obtained. At the permissive temperature of 33°C, RPE-J cells behave as an immortalized cell line. When RPE-J cells are grown on nitrocellulose filters coated with a thin layer of Matrigel in the presence of 10 8 M retinoic acid for 6 days at 33°C and then switched for 33-36 hours to the non-permissive temperature of 40°C, they acquire a differentiated polarized RPE phenotype. Under these growth conditions, RPE-J cells exhibit circumferential staining for the tight-junction protein ZO-1 and acquire a transepithelial resistance of 350 ohms cm2. Morphologically, RPE-J cells exhibit a character-istic RPE morphology with extensive apical microvilli as well as numerous dense bodies including premelanosomes and varied multilamellar structures. Ruthenium red labeling revealed the frequent basal localization of the tight junction. The cells were identified to be of rat RPE origin by their expression of the rat RPE marker RET-PE2 and their ability to phagocytose latex beads. While RPE-J cells are capable of sorting influenza and vesicular stomatitis virus to the apical and basal surfaces, respectively, the Na,K-ATPase is not polarized and the neural cell adhesion molecule, NCAM, is localized exclusively to the lateral surface. In vivo the apical surface of RPE interacts with the adjacent neural retina and the Na,K-ATPase and N-CAM are both apical; the altered polarity of these two proteins in RPE-J cells may be a consequence of the absence of apical interaction with the neural retina in culture. Previous studies of RPE have been restricted to the use of primary cultures and the RPE-J cell line should prove an excellent model system for the study of the mechanisms determining the characteristic polarity and functions of the retinal pigment epithelium.
Uptake of glutamate, not glutamine synthetase, regulates adaptation of mammalian cells to glutamine-free medium
Authors: McDermott R. H. , Butler M. 
Published: April 2021

ABSTRACT
Two cell lines (McCoy and MDCK) were studied in an attempt to understand the metabolic changes associated with adaptation to glutamine-free medium (GMEM gmate). McCoy cells assumed normal growth rates after 2-3 passages in this medium whereas MDCK cells showed no growth in GMEM + gmate.
The glutamine synthetase (GS) activity of both cell lines was elevated (up to ×9) as glutamine was depleted from normal media (GMEM gmine). The high activity of GS was maintained during McCoy cell growth in GMEM + gmate. However, there was no apparent significant difference between the two cell lines in the pattern of changes of GS activity in response to glutamine.
The cellular uptake rates of glutamine and glutamate from the medium differed significantly between the two cell lines. During the adaptation of McCoy cells to GMEM gmate, the rate of glutamate uptake doubled to a value of 0.54 nmol/min per mg cell protein whereas the maximum value for MDCK cells was considerably lower (0.04 nmol/min per mg cell protein). We propose that the difference in intrinsic ability for glutamate transport accounts for the difference in growth response between the two cell lines in the glutamine-free medium.