bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunocytochemical detection of non-histone nuclear antigens in cryosections of developing somatic embryos from Daucus carota L
Authors: SANCHEZ-PINA M. A. , KIEFT H. , SCHEL J. H.N. 
Published: April 2021

The distribution of non-histone nuclear antigens was studied during early somatic embryogenesis of carrot using nuclear antibodies specific for small nuclear RNPs (snRNPs), nuclear matrix proteins and nucleolar proteins. Immunofluorescence showed that in interphase cells snRNPs and nuclear matrix proteins were localized in the nucleus with a speckled pattern. Immunoelectron microscopy indicated that the snRNPs were irregularly distributed while the nuclear matrix proteins showed a distribution preferentially over the condensed chromatin. None were localized in the nucleolus. The nucleolar proteins were localized in both dense fibrillar and granular components while the fibrillar centres were devoid of gold particles.
The localization of these antibodies in plant cell nuclei, in a way comparable to the patterns described in animal cells, indicates the general presence of snRNPs in all eukaryotic nuclei. It further confirms the relation of the nuclear matrix proteins to plant cell proliferation events and the possible role of the nucleolar proteins in both rRNA transcription and preribosome assembly.
Enolase exists in the fluid phase of cytoplasm in 3T3 cells
Authors: Pagliaro Len , Kerr Karen , taylor D. Lansing 
Published: April 2021

ABSTRACT
We have investigated the intracellular distribution and mobility of the glycolytic enzyme enolase, using functional fluorescent analogs labeled with the succinimidyl esters of carboxyfluorescein (Flenolase) and carboxytetramethylrhodamirie (Rh-enolase). In contrast to aldolase, neither native enolase nor labeled enolase gelled filamentous actin (F-actin), as measured by falling-ball viscometry, indicating a lack of interaction between enolase and F-actin. Fluorescence redistribution after photobleaching (FRAP) measurements of the dflfusion coefficient (D) of Fl-enolase in aqueous solutions gave a value of D337,aq = 6·08×10−7cm2s-1, and no immobile fraction, consistent with a native molecular weight of 90 000. These values were not significantly dHferent with Rh-enolase, or in the presence of F-actin, 2-ρhosρhoglycerate or F-actin–aldolase gels, demonstrating that neither Fl-enolase nor Rh-enolase binds to F-actin or aldolase in vitro. FRAP measurements of Fl- and Rh-enolase microinjected into living Swiss 3T3 cells revealed spatial differences in the diffusion coefficient, but not the mobile fraction. In the perinuclear cytoplasm, we measured an apparent diffusion coefficient of l·1×10−7cm2s-1, compared to 7·1×10−8cm2s-1 in the peripheral cytoplasm, with ≈ 100% mobility of Fl- or Rh-enolase in both regions. Imaging of cells co-injected with Rh-enolase and size-fractionated FITC–dextran (FD-90) revealed that Rh-enolase entered the nucleus, while FD-90 was excluded. Ratio imaging showed a relatively high nuclear ratio of Rh-enolase/FD-90, and a uniform cytoplasmic ratio, with no indication of increased concentration of enolase around stress fibers. These data demonstrate that Rh- and Fl-enolase do not bind to F-actin in vitro, and are 100% mobile in vivo. Together with our recent finding that a significant fraction of aldolase binds to F-actin in vivo and is immobile in vivo, these data suggest a correlation between actin-binding activity and cytoplasmic mobility of glycolytic enzymes.
Type v collagen synthesis and deposition by chicken embryo corneal fibroblasts in vitro
Authors: Mclaughlin Jacqueline Shea , Linsenmayer Thomas F. , Birk David E. 
Published: April 2021

ABSTRACT
Chick embryo corneal fibroblasts were grown in culture to study the processes whereby fibroblasts regulate the deposition and organization of the collagenous, secondary stroma. The effects of an existing type I collagen substratum, cell density, and serum concentration on type V collagen synthesis were investigated. Type V collagen represented approximately 20% of the total fibrillar collagen synthesized, regardless of whether the cells were subcultured, grown on untreated or collagen-coated plastic, grown under confluent or subconfluent conditions, or grown in the presence of low (0.1%) or high (10.0%) serum concentrations. The synthesis of type V collagen remained constant at 20% of the total collagen when cells were grown in 1.0% serum, even though total collagen synthesis increased nearly twofold when compared to total synthesis in 0.1% or 10.0% serum. Immunocytochemistry with anti-collagen, type-specific monoclonal antibodies revealed a homogeneous population of cells synthesizing types I and V collagen. The fibrils deposited by cells grown in a three-dimensional collagen matrix contained a helical epitope on the type V molecule that was inaccessible unless the fibrillar structure was disrupted, mimicking the situation in situ. The production in vitro of heterotypic fibrils, with a constant l/V ratio and molecular packing mimicking the natural stroma, offers opportunities for studying in more detail this important process, which is essential for optical transparency.
Expression of oncomodulin does not lead to the transformation or immortalization of mammalian cells in vitro
Authors: Mes-Masson Anne-Marie , Masson Sylvie , Banville Denis , Chalifour Lorraine 
Published: April 2021

ABSTRACT
A recombinant plasmid (pMTONCO) containing the coding sequences for rat oncomodulin under the direction of the metallothionein promoter was constructed. pMTONCO was co-transfected with the pSV2-NEO plasmid into primary mouse kidney cells or Rat-1 cells using the calcium phosphate technique and stable transformants were isolated after selection with G418. Transcription from the metallothionein promoter was inducible with heavy metals and produced an oncomodulin-specific mRNA. The presence of oncomodulin protein in stable cell lines was verified by immune-precipitation with specific antisera. While a plasmid encoding the polyomavirus T-antigens was able to prolong the life-span of primary mouse kidney cells in culture, no equivalent activity was noted when the pMTONCO plasmid was used to transfect primary cells. When expressed in Rat-1 cells, oncomodulin did not affect the growth properties of these cells, nor did it predispose cells to higher frequencies of oncogenic transformation to a viral oncogene. We conclude that oncomodulin is neither an immortalizing nor transforming agent in vitro.
Dynamics of cytoplasmic organelles in the cell cycle of the fission yeast Schizosaccharomyces pombe: three-dimensional reconstruction from serial sections
Authors: Kanbe Toshio , Kobayashi Issei , Tanaka Kenji 
Published: April 2021

ABSTRACT
Changes in the ultrastructure of the fission yeast Schizosaccharomyces pombe during the cell division cycle were analyzed by three-dimensional reconstruction of serial section electron micrographs of freeze-substituted cells. Cytoplasmic vesicles were found at the cell ends during interphase and at the equatorial zone of cells undergoing cytokinesis. Filasomes behaved in a similar but temporally retarded way to vesicles. Microfila-ment(mf)-associated granules were found attached to the plasma membrane at the growing ends. Microfilaments were identified against the plasma membrane and adjacent to developing septa. From these observations it is suggested that mf-associated structures such as filasomes constitute dense knotsof actin network that function in localized cell wall growth by controlling the deposition of cytoplasmic vesicles. Dictyosomes occur as tubular and fenestrated cisternae with associated cytoplasmic vesicles. They were distributed uniformly in the cytoplasm and did not change significantly during the cell cycle. Changes in the three-dimensional localization of cytoplasmic microtubules and mitochondria are also described.
Human macrophage-mediated cytotoxicity of Schistosoma mansoni functional and structural features of the effector cells
Authors: Cottrell B. J. , Pye C. , Glauert A. M. , Butterworth A. E. 
Published: April 2021

ABSTRACT
Human monocytes were purified from peripheral blood and cultured in vitro on hydrophobic membranes. Such cells developed into mature tissuetype macrophages after approximately 1 week in culture. During this maturation period the macrophages developed a potent cytotoxic mechanism whereby they could kill the schistosomula of Schistosoma mansoni in standard in vitro cytotoxicity assays. Cytological and ultrastructural studies of the cells grown in vitro indicated that macrophages developed many of the classical histological and ultrastructural features of‘activated’ cells with ruffled plasma membranes and significant increases in rough endoplasmic reticulum and Golgi vesicles. Effective cytotoxicity depended upon contact of the effector cells and their parasite target. Further, experiments using metabolic inhibitors indicated that cytotoxicity was dependent upon protein synthesis. Initial results point to the macrophage factor being distinct from some of the better-characterised macrophage secretory products such as tumour necrosis factor, proteases and products of oxygen metabolism.
Suppression of malignancy in human lymphoid cell hybrids: The role of differentiation
Authors: Saltman D. , Ross J. A. , Gordon J. M. , Krajewski A. S. , Ross A. R. , Foster M. E. , Steel C. M. 
Published: April 2021

ABSTRACT
Human somatic cell hybrids were generated from fusions between a tumorigenic B-cell line, and mononuclear cells from a patient with low-grade B-cell lymphoma in which the neoplastic cells largely corresponded to cells at an intermediate stage of differentiation. The resulting hybrids and the parent cells were immunophenotyped, karyotyped and genotyped in an effort to determine whether the stage of B-cell development of the fusion partners was important in the suppression of malignancy. The majority of hybrids demonstrated suppression of tumorigenicity as measured in immunodeprived mice. Chromosome markers and immunoglobulin gene rearrangements found in the patient’s neoplastic cells were also present in several of the hybrids. One of the tumorigenic hybrids retained specific chromosomes also found in the non-tumorigenic hybrids, but demonstrated an immunophenotype and genotype possibly associated with a subset of early B-cells. In the non-tumorigenic hybrids that retained most of the chromosomes from both parents there was evidence of plasmacytoid differentiation. These results suggest that the ability of B-lymphocytes to suppress malignancy in human lymphoid hybrids may be dependent on the corresponding stage of normal B-cell ontogeny, and that suppression is associated with differentiation.
Organ maintenance of human sebaceous glands: in vitro effects of 13-cis retinoic acid and testosterone
Authors: Ridden John , Ferguson D. , Kealey Terence 
Published: April 2021

ABSTRACT
Human sebaceous glands were isolated by shearing, and maintained for 7 days either on defined medium, on medium supplemented with 3/μM-tes-tosterone or on medium supplemented with both 3 μM-testosterone and 1 μM-13-cis retinoic acid.
Freshly isolated glands retained their in vivo morphology. On maintenance, the glands retained their freshly isolated rates of cell division, but the sebocytes showed increased keratinization and there was multilayering of the peripheral undifferentiated cells. However, glands maintained in the presence of 1 μM-13-cis retinoic acid showed very little luminal keratinization and only a small degree of multilayering.
On autoradiography, freshly isolated glands retained their in vivo pattern of [methyl-3H] thymidine incorporation. Similar patterns were seen when glands were maintained for 7 days with or without testosterone. However, in the presence of both testosterone and 13-cis retinoic acid there was only slight graining.
Following 7 days maintenance the rate of lipo-genesis fell significantly. This was partially reversed by testosterone, but further inhibited by 13-cis retinoic acid. The patterns of lipids that are synthesised after a week’s maintenance are very similar to those seen in freshly isolated glands, except that the squalene:cholesterol ratio is reversibly regulated by 3 μM-testosterone and 1 μM-reti-noic acid.
Protein synthesis was maintained at the same rates as for freshly isolated glands under all conditions of maintenance. Whereas DNA synthetic rates were maintained in the presence of testosterone, they were significantly inhibited by 13-cis retinoic acid.
Glandular wet weights were retained under all conditions of maintenance, except that they were significantly reduced by 13-cis retinoic acid.
This study shows that human sebocytes continue to divide on organ maintenance, but that they do not differentiate fully. However, this provides the first demonstration that 13-cis-retinoic acid acts on human sebaceous glands directly, reducing the rate of cell division and the rate of lipogenesis, which shows that the maintained human sebaceous gland might provide a useful model for studying the effect of 13-cis retinoic acid on human sebocytes.
Steps in the morphogenesis of a polarized epithelium: II. disassembly and assembly of plasma membrane domains during reversal of epithelial cell polarity in multicellular epithelial (MDCK) cysts
Authors: Wang Allan Z. , Ojakian George K. , Nelson W. James 
Published: April 2021

ABSTRACT
A fundamental aspect in the morphogenesis of a polarized epithelium is the formation of structurally and functionally distinct apical and basal-lateral domains of the plasma membrane. The formation of these membrane domains involves the accumulation of domain-specific proteins and removal of incorrectly localized proteins. The mechanisms involved in these processes are not well understood. We have approached this problem by detailed analysis of the distribution and fate of proteins specific for different membrane domains during reversal of epithelial polarity. In the preceding paper we showed that MDCK cells form multicellular cysts comprising a closed monolayer of polarized cells. The orientation of cell polarity depends upon whether cysts are formed in suspension culture or in a collagen gel. Here, we show that, when fully developed cysts formed in suspension culture are placed in a collagen gel, polarity is rapidly reversed without cell dissociation. We show that during the process of polarity reversal, plasma membrane domains are disassembled by uptake of proteins into cytoplasmic vesicles, followed by protein degradation that probably occurs in lysosomes. The disassembly and assembly of the apical and the basal-lateral membrane domains occur in a sequential order with different kinetics. Our results provide further insights into the establishment of protein specificity of plasma membrane domains in polarized cells.
Requirement for two different cell-binding domains in fibronectin for neurite extension of neuronal derivative cells
Authors: Lewandowska Kristine , Balza Enrica , Zardi Luciano , Culp Lloyd A. 
Published: April 2021

ABSTRACT
Some neuron-derived cells, such as neuroblastoma cells, adhere and extend neurites on fibronectin (FN) substrata by processes that can be independent of binding to the Arg-Gly-Asp-Ser sequence (RGDS in FN) and independent of proteoglycan/ganglioside-binding activities of FN. Proteolytic fragments of various FNs have been used in this study to map a new adhesion-promoting domain in FNs that may be neural cell-specific. A thermolysin-generated fragment of human plasma FN (Fl 10 containing the RGDS domain) or the analagous fragment from transformed human cell FN (F120, also containing the alternately spliced extra domainb[EDb]) facilitate RGDS-independent adherence and neurite extension of human neuroblastoma cells and an Fll hybrid neuronal line (by fusion of mouse neuroblastoma cells with rat dorsal root ganglion neurons) as effectively as adherence and neurite extension on intact FN. Since neither F110 nor F120 contains sequences from the alternately spliced IIICS region of FN, neurite-promoting activity in these fragments cannot be ascribed to a recently discovered cell-binding domain in this region. Furthermore, F120 could be cleaved into two subfragments retaining virtually all the sequence of the parent fragment: F35 from the C terminus of F120 containing the RGDS domain, and F90 from the N terminus containing most of the EDb region bordering the thermolysin cleavage site. These neuronal cells could adhere but not extend neurites on substrata coated with either F35 or F90 alone while 3T3 cells could adhere only on F35. Mixtures of F35 and F90 on substrata could reconstitute some, but not nearly all, of the neurite-promoting activity of F120. Therefore, these data identify a new cell-binding domain in common sequences of FNs on the N-terminal side of EDb and demonstrate cooperativity between this RGDS-in-dependent domain and the RGDS-dependent domain for maximal differentiation of these neuron-derived cells. Several possibilities for a receptor directed to this new domain are discussed.
Cytokeratin expression in rat mesothelial cells in vitro is controlled by the extracellular matrix
Authors: Mackay Alastair M. , Tracy Russell P. , Craighead John E. 
Published: April 2021

ABSTRACT
Rat mesothelial cells co-express vimentin and the simple epithelial cytokeratins. While cytokeratins predominate in situ, under most culture conditions vimentin is the major intermediate filament protein of the cells. This loss of cytokeratin production upon culture can be partly prevented by growing mesothelial cells on a basement membrane matrix. However, the basement membrane-promoted persistence of cytokeratin synthesis is not accompanied by expression of cytokeratin G (no. 19), the major acidic cytokeratin of mesothelium in vivo.
While cells grown on plastic establish a prominent juxtanuclear assemblage of tonofilaments, those cultured on basement membrane exhibit cytokeratin filaments which are distributed throughout the cytoplasm and attach to neighboring cells at the plasma membrane. This latter pattern resembles that seen in the intact mesothelium.
Intermediate filaments are markers of cellular differentiation, but their roles are obscure. The response of cultured mesothelial cells to different growth substrata supports the hypothesis that intermediate filament synthesis is influenced by cellular contact with the extracellular matrix.
Nerve branching is induced and oriented by a small applied electric field
Authors: McCaig C. D. 
Published: April 2021

ABSTRACT
Nerve branching is controlled by intrinsic and extrinsic cues, one of which may be a small applied electric field. Lateral processes were induced by passing current through a micropipette placed at 90° to the shaft of a developing nerve. The appearance of processes was a polarised event with a large majority arising from the cathodal facing side of nerves. Whilst an electric field alone may promote branching, the presence of dimethyl sulfoxide (DMSO) or the ganglioside GM! enhanced branching of developing nerves. It is likely that an applied electric field promotes microtubule disassembly locally along the neurite shaft and that this can lead to a polarised rearrangement of the neuronal cytoskeleton. It is suggested that the use of an applied electric field in conjunction with these pharmacological agents might enhance nerve regeneration in vivo.
C-cam (cell-CAM 105) is an adhesive cell surface glycoprotein with homophilic binding properties
Authors: tingström Anders , blikstad Ingrid , aurivillius Magnus , Öbrink Björn 
Published: April 2021

ABSTRACT
C-CAM (Cell-CAM 105) is a cell surface glycoprotein that is involved in cell-cell adhesion of rat hepatocytes in vitro. To elucidate the adhesion mechanism the binding properties of purified C-CAM were investigated. Using proteins immobilized on nitrocellulose it was found that radiolabeled C-CAM bound to C-CAM but not to a variety of other proteins. Partitioning in Triton X-114 showed that C-CAM has hydrophobic properties. In accordance with this, C-CAM was effectively incorporated into phosphatidylcholine liposomes by dialysis from octylglucoside-containing solutions. The C-CAM-containing liposomes bound specifically to isolated hepatocytes. This binding was blocked by Fab fragments of antiC-CAM antibodies. Furthermore, preincubation of hepatocytes with anti-C-CAM antibodies followed by washing of the cells blocked binding of C-CAM-containing liposomes. At increasing C-CAM contents in the reconstituted liposomes a marked self-aggregation of the liposomes occurred. This aggregation was blocked by Fab fragments of anti-C-CAM antibodies and by alkaline pH. After neutralization a rapid reaggregation occurred. Neither C-CAM binding to C-CAM immobilized on nitrocellulose nor C-CAM-liposome aggregation required calcium ions. Liposomes reconstituted with C-CAM-depleted membrane glycoproteins did not self-aggregate or bind to hepatocytes. Thus, it is concluded that C-CAM can bind specifically to C-CAM in a homophilic binding reaction that does not require calcium. Accordingly, C-CAM has the potential of directly mediating cell-cell adhesion via C-CAM-C-CAM binding between adjacent cells.
Distribution of tubulin and actin through the cell division cycle of the fission yeast Schizosaccharomyces japonicus var. Versatilis: a comparison with Schizosaccharomyces pombe
Authors: Alfa Caroline E. , Hyams Jeremy S. 
Published: April 2021

ABSTRACT
Changes in the distribution of microtubules and F-actin through the cell division cycle of the fission yeast Schizosaccharomyces japonicus var. versatilis were investigated by fluorescence microscopy. The fluorescence images obtained with S. japonicus were markedly superior to those previously reported for S. pombe and revealed new details of cytoskeletal organization in this important genus. As in S. pombe, F-actin in S. japonicus was present as a concentration of ‘dots’ at the growing poles of interphase cells and as a filamentous equatorial ring directing the deposition of the cytokinetic septum. The transition between these two states occurred at late anaphase, in contrast to the situation in S. pombe where the appearance of the equatorial actin ring is tightly coupled to the early events of mitosis. During the course of cytokinesis in S. japonicus the actin ring constricted and broadened, suggesting that it is contractile. Microtubule organization in S. japonicus also revealed interesting differences from S. pombe. Whereas in S. pombe cytoplasmic microtubules are reinitiated from a pair of microtubule organizing centres (MTOCs) at the cell equator, in S. japonicus they arise by extensive microtubule growth from the spindle poles. Western blots of cell extracts enriched for tubulin by DEAE-Sephadex chromatography showed that, like S. pombe, S. japonicus contains two a-tubulins and a single β-tubulin. Whilst the al- and fi-tubulins from the two species comigrated on onedimensional polyacrylamide gels, the a2 species were electrophoretically distinct Although fundamental differences clearly exist between the two species, S. japonicus could prove to be a useful tool in basic studies of fission yeast cell biology.