bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynein from serotonin-activated cilia and flagella: extraction characteristics and distinct sites for cAMP-dependent protein phosphorylation
Authors: Stephens R. E. , Prior G.
Published: April 2021

Serotonin, an activator of adenylate cyclase, stimulates motility in molluscan gill cilia and sperm flagella. To determine and compare potential targets of cAMP action, dynein was prepared from the lateral gill cilia and sperm flagella of the mussel Mytilus edulis and the clam Spisula solidissima. In the flagella of both species, high-salt extraction removes about half of the ATPase activity, half of the and heavy chains, and the outer arms. The dynein from both species sediments at 18–20 S, contains two or three intermediate chains, and three light chains. High-salt plus detergent removes most of the remaining dynein ATPase, and heavy chains, and inner arms, also yielding a stable 18–20 S particle. In gill cilia of both species, high-salt extraction removes only 12–18% of the ATPase, up to 1/3 of the heavy chains, an equivalent amount of heavy chain, and a subset of the outer arms. The dynein sediments at 18–20 S and, in Spisula, the heavy, intermediate, and light chains precisely co-sediment. High-salt plus detergent removes another 1/3 of the heavy chains, an equivalent amount of heavy chain, and the remaining outer arms. The ATPase sediments mainly as a 13–14 S form showing considerable dissociation of co-sedimenting intermediate and light chains. The inner arms and at least half of the ciliary dynein ATPase activity remain unextractable, corresponding in mass mainly to an apparent heavy chain that is vanadate-cleavable. Cyclic AMP-dependent, calcium-independent phosphorylation takes place on specific dynein light chains in cilia but on only the dynein heavy chain in flagella. Pre-activation of the flagella prevents subsequent addition of labeled phosphate. Phosphorylation has no effect on the steady-state ATPase properties. The single phosphate added to the flagellar chain is located within the LUV1 vanadate photocleavage fragment. Considering the probable locus of the light chains and the site of the heavy chain phosphorylation, both beyond the active site and toward the base of the molecule, these distinct phosphorylations may regulate dynein action by modulating arm flexibility or interaction.
Localization of the RNA polymerase I transcription factor hUBF during the cell cycle
Authors: Roussel Pascal , André Chantal , Masson Claude , Géraud Gérard , Hernandez-Verdun Danièle
Published: April 2021

Autoantibodies directed against nucleoli that recognized a doublet of 97–94 kDa in HeLa nuclear protein extracts were identified. The two polypeptides bound equal amounts of antibody, and each was recognized by antibodies affinity purified using the other polypeptide. These antigens were localized in the secondary constriction of PtK1 cells, i.e. the nucleolar organizer regions (NORs) where ribosomal genes accumulate. They were observed in human cells in the same sites as the NOR-silver-stained proteins. The molecular mass of the antigens, their characteristics in Western blotting and their localization in nucleoli and NORs during mitosis are consistent with them being RNA polymerase I transcriptional factor, UBF. This identification was confirmed on Western blotted proteins by their identical labelling patterns, using these autoantibodies and an anti-mUBF antibody that had been previously described. We obtained definitive evidence that these autoantibodies recognize UBF by the strong positive labelling of purified hUBF (1 to 4 ng). During interphase, these autoantibodies directed against UBF labelled in a folded filament pattern as small beads that may correspond to individual transcriptional units. In electron microscopy, the antibodies were observed in the dense fibrillar component (DFC) of the nucleoli and at the periphery of the fibrillar centers (FCs). At the end of G2 phase, transcription inactivation was concomitant with the gathering of UBF at mitotic NORs. UBF was not equally distributed between NORs in human cells: some NORs scored negative (2 to 4) and the intensity of labelling of positive NORs (6 to 8) differed. In confocal microscopy, 3-dimensional analysis of mitosis indicated that UBF remained associated with NORs during all mitotic stages and that there was equal partition of UBF between the daughter cells. The relationship between proteins associated with the NORs and ribosomal gene transcription is discussed.
Composite microtubules of the axon: quantitative analysis of tyrosinated and acetylated tubulin along individual axonal microtubules
Authors: Brown Anthony , Li Yan , Slaughter Theresa , Black Mark M.
Published: April 2021

We have shown previously, using immunoelectron microscopy, that axonal microtubules (MTs) are composite, consisting of distinct domains that differ in their content of tyrosinated α-tubulin (tyr-tubulin). Here, we extend these studies using a novel preparation that permits visualization of individual axonal MTs over distances of several tens of micrometres using conventional immunofluorescence procedures. Neurons are cultured on a substratum of poly-lysine and laminin and then extracted with a MT stabilizing solution containing Triton X-100 and NaCl. These extraction conditions cause a loosening of the axonal MT array so that individual MTs separate from each other for variable distances along their length. We call this phenomenon fraying. Within the axon shaft, individual MTs can often be traced for several tens of micrometres, but fraying is most extensive in the distal 100-200 μm of the axon, where individual MTs can frequently be traced for distances of 50 to 100 μm or more to their plus ends. In some cases MTs separate completely from the axon, permitting visualization of both of their ends. Double-staining of frayed preparations with various combinations of antibodies against tyr-tubulin, acetylated α-tubulin (Actubulin) or β-tubulin, clearly revealed the composite nature of axonal MTs. Composite MTs consisted of two distinct domains, one that was relatively rich in tyrtubulin and poor in Ac-tubulin, and the other that was relatively poor in tyr-tubulin and rich in Ac-tubulin. The transition between these domains was relatively abrupt, with the tyr-tubulin-rich domain extending from the transition to the plus-end of the MT. Quantitative analyses of fluorescence intensity along individual MTs using digital image processing revealed that the relative amount of tyr-tubulin increased by approximately 800% across the transition, whereas the relative amount of Ac-tubulin decreased by approximately 60%. Within the tyr-tubulin-rich domains, the relative amount of tyr-tubulin was generally not constant, but increased from the transition to the plus-end of the MT in a nonlinear manner. We propose that the specific pattern of variation in the extent of post-translational modification along an individual MT represents a snapshot of that polymer’s growth history.
γ-Tubulin reorganization during mouse fertilization and early development
Authors: Palacios Monica J. , Joshi Harish C. , Simerly Calvin , Schatten Gerald
Published: April 2021

γ-Tubulin, a component of spindle pole bodies in fungal cells and pericentriolar material in vertebrate cells , is thought to play a role in the nucleation of microtubule growth and to define their polarity. In contrast to the adult somatic cells, microtubules are nucleated in the absence of centrioles in mammalian oocytes and early embryos. By studying acentriolar mouse oocytes and their early development following fertilization, we show that γ-Tubulin antibody crossreacts with a 50,000 Mr protein in unfertilized mouse oocytes and demonstrate that γ-Tubulin distribution is rearranged dramatically during fertilization. In unfertilized mouse oocytes, - tubulin is concentrated in the broad spindle poles of meiotic spindle (MII) and as the distinct foci which form the centers of the cytoplasmic microtubule asters (cytasters). The integrity of these γ-Tubulin foci and their cytoplasmic location is maintained during the drug- or cold-induced depolymerization of microtubules. γ-Tubulin is also found in the basal body of the mouse sperm. During fertilization, the γ-Tubulin is found at the cytastral centers as well as in the incorporated sperm basal body complex, and the γ-Tubulin foci coalesce at the perinuclear microtubule organizing regions of the two pronuclei at the first mitotic prophase. During mitosis, γ-Tubulin is found associated with broad bands that form the poles of the first mitotic spindle. By the late preimplantation stage, when newly generated centrioles have been reported to arise, tubulin remains localized at the centrosome of mitotic cells. The presence of γ-Tubulin in all microtubule organizing structures provides support for its proposed role as a component of microtubule organizing centers during the complex choreography of all microtubule assemblies.
A scale associated protein of Apedinella radians (Pedinellophyceae) and its possible role in the adhesion of surface components
Authors: Koutoulis Anthony , Ludwig Martha , Wetherbee Richard
Published: April 2021

Monoclonal antibodies have been generated against cell surface components of the unicellular phytoflagellate Apedinella radians (Pedinellophyceae). One monoclonal antibody, designated Arg 1E5/1B1, labels a scale associated protein (SAP) of 145 kDa. Immunofluorescence microscopy of whole cells as well as immunoelectron microscopy of whole cell mounts and thin sections using Arg 1E5/1B1 have shown that the SAP is located on the proximal surface of body scales and spine-scales. Its specific location suggests that the SAP may play a role in the adhesion of these surface components to the cell membrane and/or to one another. The potential of monoclonal antibody Arg 1E5/1B1 as a tool to study cell surface morphogenesis and the role of the endomembrane system in A. radians is discussed.
Three-dimensional imaging of living neurons and glia with the atomic force microscope
Authors: Parpura Vladimir , Haydon Philip G. , Henderson Eric
Published: April 2021

The atomic force microscope (AFM) was used to directly image hippocampal neurons and glia. Using chemically fixed and living cells it was possible to reconstruct three-dimensional cell structure and detect sub-cellular fea-tures such as the nucleus, mitochondria and filaments. By repeatedly scanning a single living cell we observed the movement of filaments beneath the cell membrane. Furthermore, by controlling the force applied to the scanning tip, nanosurgery was performed to selectively remove cells from the culture substratum. Thus, the atomic force microscope offers the opportunity to gain three-dimensional information about living cells and to observe the behavior of cellular components by imaging through the intact cell membrane.
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

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

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

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

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

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

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

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

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.
Cellular tensegrity: defining new rules of biological design that govern the cytoskeleton
Authors: Ingber Donald E.
Published: April 2021

“…organization exists in the living organism, and this organization is not something fundamentally mystical and unamenable to scientific attack, but rather the basic problem confronting the biologist.”

Joseph Needham (Terry Lectures, Yale University, 1936)
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

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

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

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

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

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.
Decrease in nuclear phospholipids associated with DNA replication
Authors: Maraldi Nadir M. , Santi Spartaco , Zini Nicoletta , Ognibene Andrea , Rizzoli Riccardo , Mazzotti Giovanni , Primio Roberto Di , Bareggi Renato , Bertagnolo Valeria , Pagliarini Carla , Capitani Silvano
Published: April 2021

Lipid metabolism in nuclei is very active and appears involved in the transduction of signals to the genome in response to agonists acting at the plasma membrane level. However, the precise topology of nuclear lipid metabolism and the relationship between nuclear lipids and crucial events of the cell function, such as DNA replication, have not been fully elucidated. By using a recently developed cytochemical method for detecting phospholipids inside the nucleus of intact cells at the electron microscope level, we have analyzed the changes in intranuclear phospholipids in DNA-replicating versus resting cells, which are both present in the same sample of regenerating liver after partial hepatectomy.

The pattern of DNA synthesis in replicating cells has been monitored by electron microscope immunocytochemistry after bromodeoxyuridine (BrdU) labeling. The data obtained, which allow a fine localization and a quantitative analysis of both DNA synthesis and phospholipid distribution, indicate a significant reduction in the phospholipids detectable inside the nucleus in all steps of the S phase. This could depend on an increased nuclear phospholipid hydrolysis, whose products should in turn activate some of the enzymes involved in the control of DNA replication.
Inhibition of protein kinases by 6-dimethylaminopurine accelerates the transition to interphase in activated mouse oocytes
Authors: Szöllösi Maria S. , Kubiak Jacek Z. , Debey Pascale , Pennart Henri de , Szöllösi Daniel , Maro Bernard
Published: April 2021

Mouse oocyte activation is followed by a peculiar period during which the interphase network of microtubules does not form and the chromosomes remain condensed despite the inactivation of MPF. To evaluate the role of protein phosphorylation during this period, we studied the effects of the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP) on fertilization and/or parthenogenetic activation of metaphase II-arrested mouse oocytes. 6-DMAP by itself does not induce the inactivation of histone H1 kinase in metaphase II-arrested oocytes, and does not influence the dynamics of histone H1 kinase inactivation during oocyte activation. However, 6-DMAP inhibits protein phosphorylation after oocyte activation. In addition, the phosphorylated form of some proteins disappear earlier in oocytes activated in the presence of 6-DMAP than in the activated control oocytes. This is correlated with the acceleration of some post-fertilization morphological events, such as sperm chromatin decondensation and its transient recondensation, formation of the interphase network of microtubules and pronuclear formation. In addition, numerous abnormalities could be observed: (1) the spindle rotation and polar body extrusion are inhibited; (2) the exchange of protamines into histones seems to be impaired, as judged by the morphology of DNA fibrils by electron microscopy; (3) the formation of a new nuclear envelope around the sperm chromatin proceeds prematurely, while recondensation is not yet completed. These observations suggest that the 6-DMAP-sensitive kinase(s) is (are) involved in the control of post-fertilization events such as the formation of the interphase network of microtubules, the remodelling of sperm chromatin and pronucleus formation.
Inactivation of cdc2 kinase during mitosis requires regulated and constitutive proteins in a cell-free system
Authors: Suprynowicz Frank A.
Published: April 2021

Inactivation of the cyclin-p34cdc2 protein kinase complex is a major requirement for anaphase onset and exit from mitosis. To facilitate identification of specific molecules that regulate this event in mammalian cells, I have developed a cell-free assay in which cdc2 kinase associated with a chromosomal fraction from metaphase tissue culture cells is inactivated by a cell-cycle-regulated cytosolic system. In vitro kinase inactivation requires ATP, Mg2 +and the dephosphorylation of one or more sites in the chromosomal fraction by protein phosphatase 1 and/or 2A. Cyclin B is destroyed during inactivation, while the level of p34cdc2 remains constant. Ammonium sulfate fractionation resolves the cytosolic inactivating system into at least two distinct protein components that are both required for inactivation and are differentially regulated during mitosis.
Cytoskeleton interactions in the ascus development and sporulation of Sordaria macrospora
Authors: Thompson-Coffe Catherine , Zickler Denise
Published: April 2021

The organization of actin during meiosis and sporulation in the ascus of the filamentous ascomycete Sordaria macrospora was determined by immunofluorescence without removal of the cell wall. Actin is present as a dense cortical network of microfilaments (MF) and plaques, a perinuclear shell of actin in prophase I of meiosis, and a complex array of MF involved in alignment of prespore nuclei and closure of spore cell membranes. The relationship of actin to the previously examined microtubule system of the ascus was determined by double-label immunofluorescence. The cytoskeletal inhibitors nocodazole, cytochalasin D and 2,3-butanedione monoxime were used to examine the roles of actin and myosin in ascus development. Microfilament and microtubule arrays are interdependant; disruption of one network results in abnormalities in the other. Both microfilaments and actin-myosin interaction are required for separation and migration of duplicated spindle pole bodies, septation and sporulation.
Purification of proteasomes from salmonid fish sperm and their localization along sperm flagella
Authors: Inaba Kazuo , Akazome Yasuhisa , Morisawa Masaaki
Published: April 2021

We have purified two chymotrypsin-like proteases from chum salmon sperm which have no apparent acrosome structure. Both of them were high molecular mass proteases (650 kDa and 950 kDa by gel filtration) and showed not only chymotrypsin-like activity but also trypsin-like activity. The 650 kDa protease was composed of at least eight or nine kinds of polypeptide with molecular masses ranging from 20 kDa to 30 kDa and was highly activated by low concentrations of SDS. Electron microscopy revealed that the 650 kDa protease was a ring-shaped particle. The 950 kDa protease was shown to contain at least one component that cross-reacts with an antibody against the 650 kDa protease. Finally, we revealed that the 650 kDa protease is located along the sperm flagella, by using immunofluorescence microscopy. The subunit composition, SDS-activation and molecular shape of 650 kDa salmonid protease were quite similar to those of the eukaryotic multicatalytic proteinase (proteasome), which is well known to participate in ATP-dependent degradation of ubiquitinated proteins; and, furthermore, the motility of demembranated sperm of salmonid fish is inhibited by chymotrypsin inhibitors in an ATP-dependent manner. Thus, the protease located in salmonid fish sperm flagella is a proteasome and is a strong candidate for the factor which regulates flagellar motility in an ATP-dependent manner.
Nanomelic chondrocytes synthesize, but fail to translocate, a truncated aggrecan precursor
Authors: Vertel Barbara M. , Walters Linda M. , Grier Bonnie , Maine Nancy , Goetinck Paul F.
Published: April 2021

Cartilage extracellular matrix (ECM) is composed primarily of type II collagen and large, link stabilized aggregates of hyaluronic acid and chondroitin sulfate proteoglycan (aggrecan). Maturation and function of these complex macromolecules are dependent upon sequential processing events which occur during their movements through specific subcellular compartments in the constituitive secretory pathway. Failure to complete these events successfully results in assembly of a defective ECM and may produce skeletal abnormalities. Nanomelia is a lethal genetic mutation of chickens characterized by shortened and malformed limbs. Previous biochemical studies have shown that cultured nanomelic chondrocytes synthesize a truncated aggrecan core protein precursor that disappears with time; however, the protein does not appear to be processed by the Golgi or secreted. The present study investigates the intracellular trafficking of the defective aggrecan precursor using immunofluorescence, immunoelectron microscopy and several inhibitors. Results indicate that nanomelic chondrocytes assemble an ECM that contains type II collagen, but lacks aggrecan. Instead, aggrecan precursor was localized intracellularly, within small cytoplasmic structures corresponding to extensions of the endoplasmic reticulum (ER). At no time were precursor molecules observed in the Golgi. In contrast, normal and nanomelic chondrocytes exhibited no difference in the intracellular or extracellular distribution of type II procollagen. Therefore, retention of the aggrecan precursor appears to be selective. Incubation of chondrocytes at 15°C resulted in the retention and accumulation of product in the ER. After a return to 37°C, translocation of the product to the Golgi was observed for normal, but not for nanomelic, chondrocytes, although the precursors disappeared with time. Ammonium chloride, an inhibitor of lysosomal function, had no effect on protein loss, suggesting that the precursor was removed by a non-lysosomal mechanism, possibly by ER-associated degradation. Based on these studies, we suggest that nanomelic chondrocytes are a useful model for examining cellular trafficking and sorting events and the processes by which abnormal products are targeted for retention or degradation. Further investigations should provide insight into the mechanisms underlying chondrodystrophies and other related diseases.
Expression of osteopontin mRNA by osteoclasts and osteoblasts in modelling adult human bone
Authors: Merry Karen , Dodds Robert , Littlewood Amanda , Gowen Maxine
Published: April 2021

Over recent years several non-collagenous matrix proteins of bone have been isolated and characterized. One of these proteins, osteopontin, has been shown to be synthesized by osteoblasts and deposited in the bone matrix where it is thought to bind to hydroxyapatite. However much of the functional evidence is circumstantial, and the precise function of osteopontin has not been fully elucidated.

We have used in situ hybridization techniques to investigate the expression of osteopontin mRNA in a variety of human bone tissues. Cryostat sections of human osteophyte and osteoclastoma tissue were hybridized with an antisense RNA probe for osteopontin. Sense transcripts were used as a negative control to assess non-specific binding.

There was a very distinct pattern of osteopontin mRNA expression in these tissues. Plump osteoblasts adjacent to the osteoid matrix expressed high levels of osteopontin mRNA, whilst flattened osteoblasts demonstrated weaker expression. The most striking feature of osteopontin mRNA expression was the high levels detected in osteoclasts. Osteoclasts in resorption lacunae and those distant from resorption sites both expressed osteopontin mRNA, suggesting that attachment was not a prerequisite for osteopontin expression. A population of mononuclear cells in resorption lacunae was also observed to express high levels of osteopontin mRNA. The whole population of osteoclasts in the osteoclastoma tissue expressed high levels of osteopontin mRNA, indicating that expression is not restricted to osteoclasts involved in bone resorption.

This study confirms that human osteoblasts are capable of synthesizing osteopontin. However it highlights the fact that osteoclasts may not be dependent on osteopontin present in the matrix for attachment, as they are capable of synthesizing their own osteopontin to facilitate anchorage to the bone matrix prior to resorption.
Diverse mechanisms for cell attachment to platelet thrombospondin
Authors: Adams Josephine C. , Lawler Jack
Published: April 2021

Thrombospondin-1 is a component of the extracellular matrix which is thought to play important roles in cell migration and proliferation, during embryogenesis and wound repair. To understand the basis for these activities, we are mapping the regions of the molecule with cell adhesive activity. Here, we use antagonists of specific cell binding sites, adhesion-perturbing throm-bospondin monoclonal antibodies and proteolytic fragments of platelet thrombospondin, to investigate the adhesive mechanisms used by G361 melanoma cells, human intestinal smooth muscle cells (HISM), epidermal keratinocytes and MG-63 osteosarcoma cells. When attached to the same preparations of platelet thrombospondin, HISM and MG-63 cells underwent spreading, whereas G361 cells and keratinocytes did not. Attachment of all four cell types involved the carboxy-terminal domain. The type 1 repeats and the amino-terminal heparin binding domain were important for stable attachment of G361, HISM and MG-63 cells, but were not involved in keratinocyte attachment. GRGDSP peptide caused near complete inhibition of HISM and MG-63 cell attachment, partially inhibited G361 attachment, but did not inhibit keratinocyte attachment. Attachment of HISM and MG-63 cells involved the αvα3 integrin. The integrity of the thrombospondin molecule was important for its adhesivity towards G361, HISM, and MG-63 cells, whereas keratinocytes attached to the 140 kDa tryptic fragment as effectively as they did to the intact molecule. These results show that cell attachment to platelet thrombospondin typically involves multiple binding interactions, but the exact profile of interactions is cell type specific. Usage of particular cell-binding sites does not predict whether cells will undergo spreading or not. These data may, in part, explain some of the current controversies surrounding the mechanisms of cell attachment to thrombospondin.

Abbreviations: TSP-1, thrombospondin-1; TSP-2, thrombospondin-2; TBS, Tris-buffered saline; DMEM, Dulbecco’s modified Eagle’s medium