bluuebunny/crossref_metadata_2025_split huggingface dataset:
Possible continuity of subplasmalemmal cytoplasmic network with basement membrane cord network: ultrastructural study
Authors: Inoue Sadayuki 
Published: April 2021

ABSTRACT
The ultrastructure of the subplasmalemmal cytoplasm of the cell and the associated basement membrane as well as the area of the cell-basement membrane border were observed with high resolution electron microscopy after preparation of the tissues with cryofixation or glutaraldehyde fixation followed by freeze substitution. The subplasmalemmal cytoplasm of the smooth muscle cells of rat epididymal tubules and the podocyte processes of the mouse glomerular visceral epithelium were found to be composed of a fine network of irregular anastomosing strands. This network closely resembled the previously characterized cord network of the basement membrane. The cords are known to be composed of a 1.5 to 3 nm thick core filament made up of type IV collagen which is surrounded by an irregular ‘sheath’ of other components. The strands in the subplasmalemmal network showed ultrastructural features similar to those of the cord network. Ribbon-like, 4.5 nm wide heparan sulfate proteoglycan ‘double tracks’ were previously reported to be associated with the cord network. Structures similar in size and appearance to the double tracks were also found in the subplasmalemmal network. At the cell-basement membrane border, the lamina densa of the basement membrane was in contact with the cell without the intervening space of a lamina lucida which was recently found to be an artefact caused by conventional tissue processing. Furthermore, the subplasmalemmal network appeared to be continuous, through the plasma membrane, with the cord network of the basement membrane. These results raise the possibility that the subplasmalemmal region of the cell and the associated basement membrane is occupied by a seemingly ultrastructurally unified network in spite of the fact that two such areas have been known to be composed of bichemically distinct components.
Role of the heparin binding domain of fibronectin in attachment and spreading of human bone-derived cells
Authors: Dalton B. Ann , McFarland Clive D. , Underwood P. Anne , Steele John G. 
Published: April 2021

ABSTRACT
Human bone-derived cells are known to attach and spread on surfaces which have been precoated with fibronectin, but the contributions made by specific domains of the molecule have not yet been defined. Here we refer to the osteoblast-like cells as human bone cells. We have determined the relevance of separate regions of fibronectin, particularly the heparin-binding region, for the initial attachment and spreading of these cells.
Human bone cells attached to fragments from each of the cell- and heparin-binding regions of fibronectin, but failed to attach to a fragment from the gelatin-binding region. Bovine corneal epithelial cells, which were included as an example of an alternative primary cell strain, attached to the cell-binding fragment but showed no specific shortterm attachment to the heparin or gelatin-binding fragments. Monoclonal antibody MAb17, which binds to the cell binding region of fibronectin, partially inhibited the attachment of both human bone cells and corneal epithelial cells to intact fibronectin when present at 50 µg/ml and reduced human bone cell attachment to the cell-binding region fragment of fibronectin. Monoclonal antibody, MAb 32, which binds to the heparin-binding region of fibronectin, failed to inhibit attachment of the human bone cells to fibronectin but reduced the attachment of these cells to the heparin-binding region fragment. Heparin and chondroitin sulphate were able to inhibit human bone cell attachment to the heparin-binding fragment of fibronectin but had no effect on their attachment to intact fibronectin or the cell-binding region of fibronectin.
Immunofluorescent staining and confocal microscopy showed extensive spreading and actin filament formation when human bone cells were cultured on intact fibronectin. Cells cultured on the heparin-binding fragment showed only minimal spreading coinciding with less extensive actin filament organisation. On the cell-binding fragment of fibronectin more spreading was seen than on the heparin-binding fragment but it was not as extensive as on intact fibronectin.
Taken together, these data suggest that human bone cells, unlike bovine corneal epithelial cells, have an attachment mechanism for the heparin-binding region of fibronectin. Attachment to this region is probably mediated by cell surface proteoglycans. However, interaction with the cell-binding domain is required for effective cell spreading of human bone cells on fibronectin during the first 90 minutes after seeding into culture.
RNAs radiate from gene to cytoplasm as revealed by fluorescence in situ hybridization
Authors: Dirks Roeland W. , Daniël Kurt C. , Raap Anton K. 
Published: April 2021

ABSTRACT
Genes for Epstein-Barr virus, human cytomegalovirus immediate early antigen and luciferase are abundantly transcribed in Namalwa, rat 9G and X1 cells, respectively. The EBV transcripts and HCMV-IE transcripts are extensively spliced, while in the luciferase transcript only a small intron sequence has to be spliced out. EBV transcripts are hardly localized in the cytoplasm while the luciferase and HCMV-IE transcripts are present in the cytoplasm and translated into proteins. We have correlated these characteristics with nuclear RNA distribution patterns as seen by fluorescence in situ hybridization. Transcripts of the HCMV-IE transcription unit were shown to be present in a main nuclear signal in the form of a track or elongated dot and as small nuclear RNA signals that radiate from this site towards the cytoplasm. A similar distribution pattern of small RNA signals was observed for transcripts of the luciferase gene, whereas the main nuclear signal was always observed as a dot and never as a track or elongated dot. In Namalwa cells, EBV transcripts were only present as track-like signals. The results suggest that when the extent for splicing is high, unspliced or partially spliced mRNAs begin to occupy elongated dot or track-like domains in the vicinity of the gene. When the extent of splicing is low, splicing is completed co-transcriptionally, leading to a bright dot-like signal. The presence of small nuclear spots in addition to the main signal correlates with cytoplasmic mRNA expression. The small spots most likely represent, therefore, mRNAs in transport to the cytoplasm.
Perlecan is a component of cartilage matrix and promotes chondrocyte attachment
Authors: SundarRaj Nirmala , Fite Dana , Ledbetter Steven , Chakravarti Shukti , Hassell John R. 
Published: April 2021

ABSTRACT
Aggrecan, a chondroitin/keratan sulfate-containing pro teoglycan, is a major component of cartilaginous tissues. Immunolocalization studies, using antibodies directed to perlecan, a heparan sulfate proteoglycan first detected in basement membranes, and laminin (another major component of basement membranes), indicate that perlecan and laminin are also present in the matrices of hyaline cartilage in the nasal septum, the articular surface of the bone and the growth plate of the developing bone. Consequently, we used antibodies to both aggrecan and perlecan to characterize their synthesis and secretion by primary cultures of chondrocytes derived from the rat chondrosarcoma. Chondrocytes were pulsed for 20 minutes with [35S]methionine and then chased for up to six hours. The radiolabeled perlecan and aggrecan were immunoprecipitated and analyzed by SDS-PAGE. The results show that chondrocytes synthesize precursor proteins to both proteoglycans, but that only the aggrecan precursor protein is secreted as a proteoglycan. Perlecan was also secreted but with less posttranslational modifica tions than aggrecan. Northern blot analyses of the RNAs from immortalized rat chondrocytes indicated that the major mRNA encoding for perlecan was approximately 13 kb in length, similar in size to that expressed by other cell types, which synthesize 400 kDa core protein perlecan. Analyses of the proteoglycan fractions from the extracts of bovine articular surface indicated that perlecan in this tissue contains both chondroitin and heparan sulfate side chains. Purified perlecan and laminin were found to promote attachment of immortalized rat chondrocytes in vitro. These studies indicated that perlecan, once thought to be a unique component of the basement membranes, is more widely distributed and is an important component of the cartilage matrix, where it may provide for cell adhesion to the matrix.
Myogenic conversion of mammalian fibroblasts induced by differentiating muscle cells
Authors: Salvatori G. , Lattanzi L. , Coletta M. , Aguanno S. , Vivarelli E. , Kelly R. , Ferrari G. , Harris A. J. , Mavilio F. , Molinaro M. , Cossu G. 
Published: April 2021

ABSTRACT
Somite-derived skeletal myoblasts are supposed to be the sole source of muscle fibre nuclei during pre- and postnatal development, but evidence is accumulating for unorthodox contributions to muscle fibre nuclei from other cell types. For example, in tissue culture, fibroblasts can fuse with dysgenic myoblasts and restore correct membrane function.
We report here the results of a series of experiments investigating this phenomenon and its possible mechanism. 10T1/2 cells, infected with a replication defective retrovirus encoding the bacterial enzyme β-galactosidase, fused to form β-galactosidase positive, differentiated myotubes when cocultured with differentiating uninfected C2C12 or primary myogenic cells, but this did not occur when they were cocultured with other cells such as 3T3 fibroblasts or PC12 pheochromocytoma cells. Myogenic conversion ranged from 1 to 10% of the 10T1/2 cell population and required close cell interaction between the different cells types: it was not induced by conditioned medium or extracellular matrix deposited by C2C12 cells. Myogenic conversion was also observed in vivo, after injection of similarly infected 10T1/2 cells into regenerating muscle. Conversion was seen also after coculture of uninfected 10T1/2 cells with primary chick myoblasts, thus demonstrating that it was not dependent upon viral infection and that there is no species or class barrier in this phenomenon. Primary fibroblasts, isolated from different organs of transgenic mice carrying a Lac Z marker under the control of a musclespecific promoter, restricting β-galactosidase expression to striated muscle cells, also underwent myogenic conversion, when cocultured with C2C12 myoblasts. The efficiency of this conversion varied with their embryological origin, being common in cells with a dorsal mesoderm lineage but rare in cells of ventral mesoderm origin. These experiments demonstrate that myogenic conversion is a true embryological feature of mammalian mesodermal cells. Conversion of mononucleated cells was also observed, showing that fusion is not a pre-requisite for myogenic differentiation and may indeed be a consequence of differentiation induced by short-range local signalling. We conclude that a proportion of adult cells of mesodermal origin may conserve a bi-or multi-potential state of determination throughout the life of an animal, enhancing the regenerative capacity of the tissues in which they reside.
A role for interleukin-1 in epidermal differentiation: Regulation by expression of functional versus decoy receptors
Authors: Eller Mark S. , Yaar Mina , Ostrom Kristin , Harkness Daniel D. , Gilchrest Barbara A. 
Published: April 2021

ABSTRACT
Although human epidermis contains levels of interleukin-1 (IL-1) up to 100 times higher than other tissues, the role of this cytokine in epidermal biology is unknown. Here, we show that interleukin-1 regulates the expression of mRNAs for two proteins associated with the differentiated phenotype of human keratinocytes, cellular retinoic acidbinding protein type II (CRABP II) and small, proline rich protein 1 (SPRR1). The ability of IL-1 to induce these transcripts correlates directly with keratinocyte expression of the IL-1 receptor type I (IL-1 RI) during differentiation and inversely with the expression of the type II IL-1 receptor (IL-1 RII), shown in other cell types to be a non-functional, decoy receptor. Furthermore, addition to ker-atinocyte cultures of an IL-1 RI-blocking, but not an IL-1 RII-blocking, antibody reduces the level of CRABP II and SPRR1 mRNAs in these cells. These data suggest that epidermal IL-1 functions to promote keratinocyte differentiation and that a change in the IL-1 receptor profile of these cells initiates this IL-1 response through a relative enhanced expression of functional IL-1 receptors.
Guidance of oligodendrocytes and their progenitors by substratum topography
Authors: Webb Anna , Clark Peter , Skepper Jeremy , Compston Alastair , Wood Andrew 
Published: April 2021

ABSTRACT
Oligodendrocyte progenitors arise in subventricular zones and migrate extensively during development before differentiating into mature oligodendrocytes, which myelinate nerve tracts in the central nervous system. We have used microfabricated substrata, containing periodic patterns of contours similar to those of central nervous system axons to assess the influence in vitro of substratum topography on oligodendrocytes isolated from 7 day rat optic nerve. Antiganglioside antibody A2B5 positive oligodendrocyte-type 2 astrocyte progenitors, and galactocerebroside positive and myelin basic protein positive oligodendrocytes, were highly aligned by surface contours as small as 100 nm depth and 260 nm repeat spacing. Rat optic nerve astrocytes also aligned on surface contours, but rat hippocampal and cerebellar neurons were unresponsive. Oligodendrocytes demonstrated enhanced parallel extension of their processes on narrow repeating topography in an arrangement similar to that found in the intact optic nerve. This is in marked contrast to the phenotype displayed by this cell type on planar substrata. Neither oligodendrocytes nor oligodendrocyte-type 2 astrocyte progenitors showed high-order Factin cytoskeletal networks; thus their alignment on gratings is unlikely to result from deformation of actin cables and focal contacts. In contrast, aligned astrocytes showed striking arrangements of actin stress fibres. These results establish glial cells as potentially the most topographically sensitive cell types within the central nervous system. Furthermore, the topographical pattern inducing maximal alignment of oligodendrocyte lineage cells corresponds to the diameters of single axons within the 7 day optic nerve. Thus the migration of oligodendrocyte-type 2 astrocyte progenitors and axonal ensheathment by oligodendrocytes may be guided by axonal topography within the developing nerve.
Microtubules released from the neuronal centrosome are transported into the axon
Authors: Ahmad Fridoon J. , Baas Peter W. 
Published: April 2021

ABSTRACT
There is controversy concerning the source of new microtubules required for the development of neuronal axons. We have proposed that microtubules are released from the centrosome within the cell body of the neuron and are then translocated into the axon to support its growth. To investigate this possibility, we have developed an experimental regime that permits us to determine the fate of a small population of microtubules nucleated at the neuronal centro-some. Microtubules within cultured sympathetic neurons were depolymerized with the anti-microtubule drug noco-dazole, after which the drug was removed. Microtubules rapidly and specifically reassembled from the centrosome within three minutes of nocodazole removal. At this point, low levels of vinblastine, another anti-microtubule drug, were added to the culture to inhibit further microtubule assembly while not substantially depolymerizing the small population of microtubules that had already assembled at the centrosome. Within minutes, released microtubules were apparent in the cytoplasm, and many of these had already translocated to the cell periphery by ten minutes. By one hour, virtually all of the microtubules had been released from the centrosome and were concentrated at the cell periphery. With increasing time, these microtubules appeared within and progressively farther down developing axons. Nonneuronal cells within the culture also reassembled microtubules at the centrosome, but only a small portion of these microtubules were released. These observations indicate that microtubules are released from the neuronal centrosome and transported into growing axons, and that microtubule release and relocation from the centrosome are especially active in neurons compared to nonneuronal cells.
Galectin-3 expression and effects on cyst enlargement and tubulogenesis in kidney epithelial MDCK cells cultured in three-dimensional matrices in vitro
Authors: Bao Qi , Hughes R. Colin 
Published: April 2021

ABSTRACT
Galectin-3 is a member of a closely related family of β-galactoside-binding soluble proteins found in many vertebrate epithelial and myeloid cell types. The developmentally regulated presence of galectin-3 in tissues, for example kidney, and an affinity for many cell-surface and matrix glycoproteins indicate its importance in extracellular biological processes. Since a polarised expression and secretion of galectin-3 was observed in monolayer-cultured MDCK cells, an understanding of the secretion and distribution of this lectin in a three-dimensional in vitro model would help to uncover its role(s) in the interplay between cell-surface adhesion molecules and extracellular matrix components occurring during cell aggregation and polarisation in tissue formation. In this study, the cellular distribution and secretion of galectin-3 were examined in MDCK cells cultured within a gel matrix. MDCK cells were cultured within type I collagen or Matrigel to obtain multicellular cysts, and tubule formation was induced in collagen gels with hepatocyte growth factor. Immunofluo-rescent staining of these structures using antibodies against galectin-3 and other cell-surface domain markers was carried out either in situ or on cryosections and was visu-alised by confocal and conventional epifluorescence microscopy. Our results show that MDCK cells suspended in hydrated collagen gels or Matrigel exhibit differential and polarised galectin-3 expression on the baso-lateral surface domains of cells lining the cysts. The lectin is colocalised with laminin on the basal surface. In tubuleforming cysts, galectin-3 is excluded from the initial spikes and the progressing tips of the tubules although its baso-lateral expression on the cyst body remains. Galectin-3 added exogenously to cultures, as well as antibodies against laminin subunits and integrin β1 subunit, exerted an inhibitory effect on cyst enlargement of MDCK cells in 3D Matrigel while galectin-3-specific antibodies could promote this process. The results suggest that galectin-3 exerts its effect on MDCK cells in a three-dimensional environment through modulation of both cell-cell and cell-substratum adhesions, and the interplay between these adhesions is important in the growth of multicellular aggregates and extensions occurring during normal kidney tubulogenesis.
Immunocytochemical localization of β-COP to the ER-Golgi boundary and the TGN
Authors: Griffiths Gareth , Pepperkok Rainer , Locker Jacomine Krijnse , Kreis Thomas E. 
Published: April 2021

ABSTRACT
Recent data strongly suggest that the coatomer (COP) complex is involved in membrane transport between the ER and Golgi complex. This vesicular coat has been implicated in ER to Golgi, in intra Golgi as well as in Golgi to ER traffic. In this study we present a detailed immunocytochemical analysis of the distribution of β-COP in different tissue culture cells. Our results extend previous studies by showing, using electron microscopy, that β-COP accumulates on vesicular profiles and buds in the intermediate compartment (IC) under conditions that block ER to Golgi transport (15°C). Importantly, under these conditions β-COP co-localizes on these structures with a passenger protein, the membrane glycoprotein of vesicular stomatis virus (ts-O45-G). Furthermore, quantitative immunofluorescence microscopy of cells with ts-045-G accumulated in the ER, IC and trans-Golgi network, shifted briefly to the permissive temperature, showed that β-COP was associated with many of the putative transport intermediates containing the viral glycoprotein which is in transit between the ER/IC and the cis-Golgi. The simplest interpretation of these data is that COP-coated vesicles are involved in anterograde transport of ts-045-G from the IC to the Golgi complex. Since many putative COP vesicles lacked the G protein following release of the 15°C block this pool could be involved in retrograde transport. We also show that β-COP is present on the membranes of the transGolgi network. However, in contrast to the ER-Golgi boundary, we could find no convincing evidence that this pool of β-COP is associated with buds or trans-Golgi network-derived transport vesicles.
Inactivation of Cdc2 increases the level of apoptosis induced by DNA damage
Authors: Ongkeko Weg , Ferguson David J. P. , Harris Adrian L. , Norbury Chris 
Published: April 2021

ABSTRACT
A number of lines of evidence have suggested a possible involvement of the mitosis-promoting protein kinase Cdc2 in the process of apoptotic cell death, and one recent study concluded that premature activation of Cdc2 is required for apoptosis. Here we have used a temperature-sensitive murine Cdc2 mutant cell line and Cdc2 inhibitor compounds to study the effect of inhibition of this protein kinase on apoptosis induced by DNA-damaging drugs. Inhibition of Cdc2 activity before or during exposure to DNA strand break-inducing drugs had the effect of increasing the level of subsequent apoptosis, as assessed by electron microscopy and flow cytometry. We conclude that, far from being required for cell death, a form of mammalian Cdc2 suppresses apoptosis induced by DNA damage. This form of Cdc2 appears to be active in G2-arrested cells and is therefore presumably distinct from the mitosis-promoting Cdc2-cyclin B heterodimer.
Identification of the nuclear localization signal of mouse DNA primase: nuclear transport of p46 subunit is facilitated by interaction with p54 subunit
Authors: Mizuno Takeshi , Okamoto Tomoko , Yokoi Masayuki , Izumi Masako , Kobayashi Akio , Hachiya Takahisa , Tamai Katsuyuki , Inoue Tadashi , Hanaoka Fumio 
Published: April 2021

ABSTRACT
DNA polymerase α-primase is a replication enzyme necessary for DNA replication in all eukaryotes. Mouse DNA primase is composed of two subunits: a 46 kDa protein (p46), which is the catalytic subunit capable of RNA primer synthesis, and a 54 kDa protein (p54), whose physiological role is not clear. To understand the structure-function relationship of DNA primase, we set out to characterize these two subunits individually or in combination using a cDNA expression system in mammalian cultured cells, and determined the subcellular distribution of ectopically expressed DNA primase. The p54 expressed in COS-1 cells after transfection was predominantly localized in the nucleus, whereas p46 was retained in the cytoplasm as shown by indirect immunofluorescence analysis. Using several mutant proteins with deletions or substitutions as well as chimeric constructs, we identified the nuclear localization signal of p54 as RIRKKLR, encoded near the amino terminus (residues 6-12). Furthermore, co-expression of both p46 and p54 subunits markedly altered the subcellular distribution of p46; co-expressed p46 was transported into the nucleus as efficiently as p54. These results demonstrate that p54 has a nuclear localization signal and is able to be translocated into the nucleus independently of DNA polymerase α subunits. In contrast, p46 lacks a nuclear localization signal, and its nuclear translocation is facilitated by interaction with p54. We present here first evidence for a novel role of p54 in the nuclear translocation process, and a piggy-back binding transport mechanism of mouse DNA primase.