bluuebunny/crossref_metadata_2025_split huggingface dataset:
Patterns in Dictyostelium discoideum: the role of myosin II in the transition from the unicellular to the multicellular phase
Authors: Eliott Susannah , Joss Gregory H. , Spudich Annamma , Williams Keith L. 
Published: April 2021

ABSTRACT
Dictyostelium discoideum amoebae which lack the myosin II gene are motile and aggregate to form rudimentary mounds, but do not undergo further morphological development (Manstein et al., 1989). Here we use scanning electron microscopy, light microscopy, immunofluorescence and computer analysis of timelapse video films to study how D. discoideum myosin null cells of strains HS2205 and HS2206 aggregate. Myosin null cells are sufficiently coordinated in their movements to form two-dimensional aggregation streams, although mutant cells within streams lack the elongated shape and parallel orientation of wild-type strains. In the wild-type, cell movements are coordinated, cells usually joining streams that spiral inwards and upwards as the mound extends into the standing papilla. In the aggregates of mutant strains, cell movements are chaotic, only occasionally forming short-term spirals that rotate at less than half the speed of wild-type spirals and frequently change direction. Unlike the situation in the wild-type where spirals continue with mound elongation, cells within the mutant mound eventually cease translocation altogether as the terminal shape of the mound is reached and only intracellular particle movement is observed. Scanning electron micrographs show that the surface of the wild-type mound consists of flattened cells which fit neatly together. The myosin null cell mound has an uneven surface, the orientation of the cells is chaotic and no tip is formed. This is consistent with the results of synergy experiments in which myosin null cells were absent from the tips of chimeric HS2205/AX2 slugs and pre-culminates. Immunofluorescence microscopy using prespore and spore cell markers reveals that a prestalk/prespore pattern forms within the mutant mound but that terminal spore differentiation is incomplete. These results are discussed in relation to the role of myosin II in aggregation and morphogenesis.
Fibroblasts expressing mouse c locus tyrosinase produce an authentic enzyme and synthesise phaeomelanin
Authors: Winder Alison J. , Wittbjer Anna , Rosengren Evald , Rorsman Hans 
Published: April 2021

ABSTRACT
Recent advances in the study of the molecular biology of mouse pigmentation have led to the discovery of a family of proteins involved in the control of melanin synthesis. It has been confirmed that the product of the mouse c (albino) locus is the key melanogenic enzyme tyrosinase, but study of its function and regulation have been hampered by the presence of closely related proteins within melanin-synthesising cells. To overcome these problems, we have established lines of mouse fibroblasts expressing the c locus mouse tyrosinase. Here we describe characterisation of the tyrosinase synthesised by these cells and demonstrate considerable similarity between the expressed tyrosinase and the native enzyme. The expressed tyrosinase is proteolytically cleaved to produce membrane-bound and soluble forms of the expected molecular mass and is rich in N-linked carbohydrate, suggesting that melanocytic differentiation is not a prerequisite for post-translational modification of the protein. The expressed enzyme has tyrosinase activity, but not catalase or dopachrome tautomerase activity, confirming that it is an authentic tyrosinase. Transfected fibroblasts expressing tyrosinase are shown to share several physiological characteristics with melanoma cell lines, including increased pigmentation and tyrosinase activity in response to increased cell density. Since tyrosinase is expressed under a heterologous promoter, these shared characteristics probably reflect translational or post-translational controls that operate in both non-melanocytic and melanocytic cell types. We demonstrate that pigmented fibroblasts contain the melanin synthesis intermediates 5-S-cysteinyldopa and 5-S-glutathionyldopa, and produce a phaeomelanin-like pigment, but do not contain detectable eumelanin. Expression of tyrosinase is therefore sufficient for the synthesis of a form of melanin pigment in fibroblasts.
Cell membrane-associated proteoglycans mediate extramedullar myeloid proliferation in granulomatous inflammatory reactions to schistosome eggs
Authors: Alvarez-Silv Márcio , da Silva Luiz-Cláudio F. , Borojevic Radovan 
Published: April 2021

ABSTRACT
In chronic murine schistosomiasis, extramedullar myelopoiesis was observed, with proliferation of myeloid cells in liver parenchyma and in periovular granulomas. We have studied the question of whether cells obtained from granulomatous connective tissue may act as myelopoietic stroma, supporting long-term myeloid proliferation. Primary cell lines (GR) were obtained in vitro from periovular granulomas, induced in mouse livers by Schistosoma mansoni infection. These cells were characterized as myofibroblasts, and represent liver connective tissue cells involved in fibro-granulomatous reactions. They were able to sustain survival and proliferation of the multipotent myeloid cell lines FDC-P1 and DA-1 (dependent on interleukin-3 and/or granulo-cyte-macrophage colony stimulating factor, GM-CSF) without the addition of exogenous growth factors. This stimulation was dependent upon myeloid cell attachment to the GR cell layer; GR cell-conditioned medium had no activity. Primary murine skin fibroblasts could not sustain myelopoiesis. The endogenous growth-factor was identified as GM-CSF by neutralization assays with monoclonal antibodies. The stimulation of myelopoiesis occurred also when GR cells had been fixed with glutardialdehyde. The observed stimulatory activity was dependent upon heparan sulphate proteoglycans (HSPGs) associated with GR cell membranes. It could be dislodged from the cell layer with heparin or a high salt buffer. Our results indicate a molecular interaction between endogenous growth-factor and HSPGs; this interaction may be responsible for the stabilization and presentation of growth factors in myelopoietic stromas, mediating extramedullar proliferation of myeloid cells in periovular granulomas.
A dynamic continuum of pleiomorphic tubules and vacuoles in growing hyphae of a fungus
Authors: Shepherd V. A. , Orlovich D. A. , Ashford A. E. 
Published: April 2021

ABSTRACT
The vacuole system in growing hyphal tips of Pisolithus tinctorius is a dynamic continuum of vacuoles and extensible tubular elements. The system varies from a tubular reticulum with few vacuoles across a spectrum of intermediate forms to clusters of vacuoles with few tubules. Spherical vacuoles interconnected in clusters are situated at intervals along the hyphal tip and are transiently linked by tubules that extend from a vacuole in one cluster and fuse with that of another. Extension and retraction of the tubules is independent of cytoplasmic streaming, can occur in either direction, and covers distances as great as 60 μm. The tubules pulsate and peristalsis-like movements transfer globules of material along them between the vacuoles in different clusters. The tubules also generate vacuoles. The tubular system has the potential for intracellular transport of solutes in the hyphal tips without concomitant transfer of large amounts of membrane. This contrasts with models of intracellular transport via vesicles, where the ratio of membrane transferred to internal content is very much higher. The system has many features in common with tubular endosomal and lysosomal systems in cultured animal cells.
Casein kinase II copurifies with yeast DNA topoisomerase II and re-activates the dephosphorylated enzyme
Authors: Cardenas Maria E. , Walter Robin , Hanna David , Gasser Susan M. 
Published: April 2021

ABSTRACT
Mitotic division in yeast requires the activity of topoisomerase II, a DNA topology modifying enzyme that is able to disentangle sister chromatids after DNA replication. Previous work has shown that topoisomerase II is a phosphoprotein in intact yeast cells. We show here that when dephosphorylated in vitro, topoisomerase II is unable to cleave or decatenate kinetoplast DNA. An efficient kinase activity that modifies topoisomerase II on seven major sites was found to copurify with the enzyme purified from yeast. Characterization of this kinase, analysis of phosphotryptic peptides, and studies with a yeast mutant deficient in casein kinase II, indicate that the copurifying kinase is casein kinase II (CKII). Topoisomerase II itself has no self-phosphorylating activity. Modification of topoisomerase II by the copurifying kinase is sufficient to restore decatenation activity after dephosphorylation by alkaline phosphatase. The CKII target sites have been mapped to multiple serine and threonine residues on 4 tryptic fragments within the C-terminal 350 amino acids of yeast topoisomerase II. These results are consistent with a model in which the C-terminal domain of topoisomerase II is a negative regulatory domain that is neutralized by phosphorylation.
Presence of a nuclear lamina in pachytene spermatocytes of the rat
Authors: Vester Bettina , Smith Avril , Krohne Georg , Benavente Ricardo 
Published: April 2021

ABSTRACT
The nuclear lamina is a karyoskeletal structure located at the periphery of cell nuclei. The major constituents are the lamins, which belong to the evolutionarily conserved multigene family of intermediate filament proteins. Lamins show a conspicuous cell type-specific expression pattern. The majoritiy of somatic cells of vertebrates express A-type (lamins A and C) as well as B-type (lamins B1 and B2) lamins. Although a lamina structure has been demonstrated to be a ubiquitous component of somatic nuclei its existence in certain meiotic stages during spermatogenesis has been a matter of debate. In this study, we investigated the expression of lamins in rat spermatogenic cells using immunological and protein-chemical methods. We report on the presence of a nuclear lamina structure in rat pachytene spermatocytes. With the aid of a novel broad-reacting lamin antibody we have demonstrated the expression of a protein that is closely related, if not identical, to lamin B1.
Differential replication capacities of G1 and S-phase extracts from sea urchin eggs
Authors: Zhang Hui , Ruderman Joan V. 
Published: April 2021

ABSTRACT
Sea urchin eggs are arrested in G1 of the first mitotic cell cycle. Fertilization triggers release from G1 arrest and the onset of DNA synthesis about 20 minutes later, even when protein synthesis is blocked. Here we describe extracts from eggs and S-phase embryos that reproduce this stage-specific pattern of DNA synthesis. Fertilized egg extracts formed nuclear membranes around decondensed Xenopus sperm chromatin whereas unfertilized egg extracts did not. Aphidicolin-sensitive deoxynucleotide incorporation was high in extracts of fertilized S-phase eggs and low in those of unfertilized eggs. In contrast, single-stranded DNA templates directed high rates of incorporation in both unfertilized and fertilized egg extracts, suggesting that the stage-specific activities in nuclear DNA synthesis is restricted to initiation on double-stranded DNA. Mixing experi-ments showed that unfertilized eggs do not contain a dominant inhibitor of replication, nor does fertilization induce the appearance of a soluble, dominant activator.
Molecular cloning of a human homologue of Drosophila heterochromatin protein HP1 using anti-centromere autoantibodies with anti-chromo specificity
Authors: Saunders William S. , Chue Calvin , Goebl Mark , Craig Carolyn , Clark Robert F. , Powers James A. , Eissenberg Joel C. , Elgin Sarah C. R. , Rothfield Naomi F. , Earnshaw William C. 
Published: April 2021

ABSTRACT
We have identified a novel autoantibody specificity in scleroderma that we term anti-chromo. These antibodies recognize several chromosomal antigens with apparent molecular mass of between 23 and 25 kDa, as determined by immunoblots. Anti-chromo autoantibodies occur in 10-15% of sera from patients with anti-cen-tromere antibodies (ACA). We used anti-chromo antibodies to screen a human expression library and obtained cDNA clones encoding a 25 kDa chromosomal autoantigen. DNA sequence analysis reveals this protein to be a human homologue of HP1, a heterochromatin protein of Drosophila melanogaster. We designate our cloned protein HP1Hsa. Epitope mapping experiments using both human and Drosophila HP1 reveal that antichromo antibodies target a region at the amino terminus of the protein. This region contains a conserved motif, the chromo domain (or HP1/Pc box), first recognized by comparison of Drosophila HP1 with the Poly - comb gene product. Both proteins are thought to play a role in creating chromatin structures in which gene expression is suppressed. Anti-chromo thus defines a novel type of autoantibody that recognizes a conserved structural motif found on a number of chromosomal proteins.
A stress-inducible 40 kDa protein (hsp40): purification by modified two-dimensional gel electrophoresis and co-localization with hsc70(p73) in heat-shocked HeLa cells
Authors: Hattori Hirotomo , Kaneda Toshio , Lokeshwar Balakrishna , Laszlo Andrei , Ohtsuka Kenzo 
Published: April 2021

ABSTRACT
We have previously reported that a novel 40 kDa protein is induced by heat shock and several environmental stresses in mammalian and avian cells and that the N-terminal amino acid sequence of this 40 kDa protein has homology with the bacterial DnaJ heat-shock protein. We have purified this protein (40 kDa heat-shock protein, hsp40) from HeLa cells by modified two-dimensional gel electrophoresis and generated a polyclonal antibody against hsp40. This antibody was highly specific for human hsp40 and cross-reacted weakly with rat and Chinese hamster hsp40. Indirect immunofluorescence revealed that the hsp40 in HeLa cells accumulates in the nucleus, especially in the nucleolus, during heat shock and returns to the cytoplasm during the recovery period. The kinetics of the accumulation in the nucleoli and subsequent return to the cytoplasm of hsp40 was similar to that of hsp70. In addition, hsp40 was co-localized with hsc70(p73) in heat-shocked HeLa cells as demonstrated by double immunofluorescence staining. These results suggest that hsp40 (a DnaJ homologue) and hsp70 (a DnaK homologue) may act in concert to repair (refold) denatured proteins and protein aggregates in the nuclei and nucleoli of heat-shocked HeLa cells.
Spatial organization of microfilaments and vitronectin receptor,αvβ3, in osteoclasts: A study using confocal laser scanning microscopy
Authors: Lakkakorpi P. T. , Helfrich M. H. , Horton M. A. , Väänänen H. K. 
Published: April 2021

ABSTRACT
The primary function of the osteoclast is that of the major cell mediating bone resorption. They are actively migrating cells but during resorption they polarize to form a specialized tight attachment structure, the sealing zone, adjacent to the mineralized bone matrix. The processes of adhesion to, and migration on, bone involves cell adhesion molecules, integrins, interacting with their ligands in bone. We have used confocal microscopy to analyse, in rat osteoclasts cultured on bone and glass substrata, the distribution of vitronectin receptor, the major integrin of osteoclasts, and cytoskeletal proteins that it may be linked to. Double staining for F-actin and vinculin, and for vinculin with talin, revealed that cytoskeletal organization differs at various activation states of osteoclasts. Microfilament structures were flat, of 1-5 m size, and concentrated near the bone surface. The vitronectin receptor was localized both in the basolateral membrane (away from the bone surface) and in the ruffled border (adjacent to bone) in osteoclasts cultured on bone, but was detected mainly in the basolateral membrane when cultured on glass. The vitronectin receptor appeared to be condensed on small microvilli-like projections on the basolateral membrane of osteoclasts on either bone or glass and may provide a route for alternative signalling pathways to modify osteoclast behaviour, other than by influencing cell adhesion directly. The leading edges of migrating osteoclasts, and the attachment structure, a broad vinculin band, which forms before bone resorption, also expressed vitronectin receptor, particularly when the antibody against the αv subunit was used. F-actin-stained central part of the sealing zone, which is between a vinculin “double circle”, failed to be stained with any of the vitronectin receptor antibodies used. These results suggest that the vitronectin receptor is involved in migration and the initial attachment of osteoclasts to the bone surface, but that at the sealing zone there is some other, thus far unknown, mechanism which mediates the tight attachment of the osteoclast cell membrane to bone. Differences in staining for αv and were β3 observed, expression of the two chains of vitronectin receptor not being totally coincident. Whilst this could be caused by immunochemical differences between the antibodies used, the finding also implies that novel integrin dimer combinations may exist in osteoclasts.
Functional properties of proximal tubule cell lines derived from transgenic mice harboring L-pyruvate kinase-SV40 (T) antigen hybrid gene
Authors: Lacave R. , Bens M. , Cartier N. , Vallet V. , Robine S. , Pringault E. , Kahn A. , Vandewalle A. 
Published: April 2021

ABSTRACT
This study describes the functional characterization of two cell lines derived from the proximal convoluted (PKSV-PCT cells) and proximal straight (PKSV-PR) tubules microdissected out from kidneys of transgenic mice harboring the simian virus 40 (SV40) large T and small t antigens placed under the control of the rat L-type pyruvate kinase (L-PK) 5 regulatory sequence. Both cell lines exhibited cellular cyclic AMP stimulated by parathormone (PTH) and calcitonin (CT) and a sodium-dependent glucose transporter. Uptake of the fluid-phase marker [3H]inulin showed that both cell lines grown on filters exhibited biphasic apical and basolateral endocytic rates. Results from Northern blot analysis indicate that the expression of the T antigen gene (Tag) is dependent on the concentration of D-glucose in the medium and show that the L-PK construct has maintained its capacity for upor down-regulation by carbohydrates. Replacement of D-glucose by neoglucogenic substrates (lactate, oxaloacetate) blunted the expression of Tag transcripts and induced arrest of cell growth. Compared to cell grown in D-glucoseenriched medium, the hormonal sensitivities to PTH and CT and the sodium-dependent glucose uptake were unchanged whereas quiescent cells exhibited increased hydrolase content. Thus the proximal function has been preserved in these cultured cells derived from tissuespecific targeted oncogenesis in transgenic mice. As the expression of Tag transcripts is controlled by D-glucose, the structural and physiological characteristics of these cell lines can be studied in either quiescent or active growth conditions.
Stratification-related expression of isoforms of the desmosomal cadherins in human epidermis
Authors: Arnemann Joachim , Sullivan Kate H. , Magee Anthony I. , King Ian A. , Buxton Roger S. 
Published: April 2021

ABSTRACT
Desmosomal junctions are abundant in epidermis and contain two classes of transmembrane glycoprotein, the desmocollins and the desmogleins, which are members of the cadherin superfamily of Ca2+-dependent cell adhesion molecules. The desmocollin subfamily includes DGIV/V and DGII/III while the desmoglein subfamily includes DGI, HDGC and the autoantigen of the blistering skin disease pemphigus vulgaris (PVA). There are also several non-glycosylated proteins, including the desmoplakins and plakoglobin, present in the desmosomal plaque, which forms a link between the glycoproteins and the cytokeratin intermediate filaments. To provide a picture of the expression of the desmosomal genes and their products in epidermis, we have used in situ hybridisation and immunofluorescence staining on sections of human foreskin. We find that, as expected, desmoplakin DPI/II and plakoglobin are expressed throughout the epidermis, gradually accumulating during differentiation, which probably reflects the increased numbers of desmosomes. In contrast, while keratin 14 and the hemidesmosomal component bullous pemphigoid antigen I (BPAGI) are basal-specific, desmocollin DGIV/V is expressed only in the upper spinous/granular layers of the epidermis, whereas DGII/III expression is enriched in the basal layers. Amongst the desmogleins, expression of DGI appears similar to desmoplakin and plakoglobin; PVA is more prevalent in the lower spinous layers, whereas HDGC expression is detected basally but not suprabasally. The major desmosomal cadherin transcripts are desmocollin DGIV/V and desmoglein DGI. The resultant changes in desmosomal composition and structure may reflect the maturation of desmosomes, presumably being related to the need for changes in cell adhesion during stratification, terminal differentiation, and desquamation, and point to the desmosome being a key player in epidermal differentiation.
Immunohistochemical localization of adherens junction components in blood-brain barrier microvessels of the rat
Authors: Schulze Charlotte , Firth J. Anthony 
Published: April 2021

ABSTRACT
The morphology and molecular composition of inter-cellular adherens junctions have most frequently been described in epithelial cells and the fascia adhaerens of the intercalated disc. A group of cytoplasmic molecules is known to be associated with adherens junctions. The intercellular bond is mediated by cadherins which bridge the cells by homophilic binding. Recently, endothelial cells have also been shown to form intercellular junctions of the adherens-type. However, they are morphologically less distinct and little is known about their molecular components.
In this study we report the localization of some adherens junction components in intact microvessels of the blood-brain barrier in the rat. We used antibodies raised against α -actinin, vinculin, zyxin, cadherin (antipan-cadherin antibody) and A-CAM (N-cadherin) in immunohistochemical experiments at light and electron microscopical levels. Microvessel walls reacted positively for all antigens throughout postnatal development. All antigens were localised, though not necessarily exclusively, to interendothelial junctions. At the ultrastructural level, pan-cadherin reactivity was present throughout the entire length of the cleft. These results could mean that in blood-brain barrier endothelial cells the complex tight junction is embedded in an adherens junction which occupies the entire length of the cleft.