bluuebunny/crossref_metadata_2025_split huggingface dataset:
A novel phosphoglucomutase-related protein is concentrated in adherens junctions of muscle and nonmuscle cells
Authors: Belkin Alexey M. , Klimanskaya Irina V. , Lukashev Matvey E. , Lilley Kathryn , Critchley David R. , Koteliansky Victor E. 
Published: April 2021

ABSTRACT
Using five monoclonal antibodies raised against a human uterine smooth muscle extract, we have identified a novel antigen which runs as a closely spaced doublet in SDS-gels. The proteins (60/63 kDa) co-purify, are present in a 1:1 ratio as judged by Coomassie Blue staining, and are immunologically closely related, if not identical. No N-terminal sequence could be obtained from a mixture of the 60/63 kDa proteins, but the sequence of four polypeptides liberated by V8 protease or cyanogen bromide cleavage showed that the proteins are closely related to the glycolytic enzyme phosphoglucomutase type 1. Affinity-purified poly-clonal antibodies and three different monoclonal antibod-ies to the 60/63 kDa proteins cross-reacted with rabbit skeletal muscle phosphoglucomutase type 1, whilst two additional monoclonal antibodies were specific for the 60/63 kDa proteins. Peptide maps of the 60/63 kDa proteins and phosphoglucomutase 1 are markedly different, and the purified proteins have no detectable phosphoglucomutase activity.
Staining of cultured smooth muscle cells and fibroblasts with antibodies to 60/63 kDa proteins showed that the antigen is concentrated in focal contacts at the ends of actin bundles and is also associated with actin filaments. About 60% of the cellular 60/63 kDa proteins were found in the detergent-insoluble fraction, suggesting a physical associa-tion with the cytoskeleton. The highest levels of protein immunoreactivity were found in muscles. The antigen is concentrated in muscle adherens junctions, including smooth muscle dense plaques, cardiomyocyte intercalated disks, and striated muscle myotendinous junctions. Among epithelial cells, the 63 kDa isoform of the protein was found only in cultured keratinocytes where immunofluorescent staining was localized in cell-to-cell adherens junctions. Expression of the 60/63 kDa proteins in vascular smooth muscle cells is developmentally regulated and correlates with the differentiated contractile phenotype of these cells.
Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate beads
Authors: Häuselmann Hans J. , Fernandes Russell J. , Mok Su S. , Schmid Thomas M. , Block Joel A. , Aydelotte Margaret B. , Kuettner Klaus E. , Thonar Eugene J.-M. A. 
Published: April 2021

ABSTRACT
Articular chondrocytes embedded in alginate gel produce de novo a matrix rich in collagens and proteoglycans. A major advantage of this culture system is that the cells can be recovered by chelating the calcium, which otherwise maintains the alginate in its gel state. Chondrocytes thus released are surrounded by tightly bound cell-associated matrix, which seems to correspond to the pericellular and territorial matrices identified in cartilage by electron microscopy. The cells and their associated matrix can be easily separated by mild centrifugation from more soluble matrix components derived principally from the ‘interter-ritorial’ matrix. This new cell culture system thus makes it possible to study the assembly and turnover of molecules present in two distinct matrix pools. Importantly, a signif-icant proportion of the aggrecan molecules in each of these two pools can be extracted using a non-denaturing solvent, thereby making possible studies of the metabolism and turnover of native proteoglycan aggregates. We show in this report that chondrocytes isolated from the full depth of adult bovine articular cartilage and maintained for 8 months in alginate gel are still metabolically active and continue to synthesize cartilage-specific type II collagen and aggrecan. The cells did not synthesize large amounts of type I collagen or of the small nonaggregating proteo-glycans as usually occurs when chondrocytes lose their phe-notypic stability. After this extended period of time in culture, the cells were present as two populations exhibit-ing differences in size, shape and amount of extracellular matrix surrounding them. The first population was found only near the surface of the bead: these cells were flattened and surrounded by a matrix sparse in proteoglycans and collagen fibrils. The second population was found through-out the remaining depth of the bead: the cells were more round and almost always surrounded by a basket-like meshwork consisting of densely packed fibrils running tan-gential to the surface.
Selective modulation of the interaction of α7β1 integrin with fibronectin and laminin by l-14 lectin during skeletal muscle differentiation
Authors: Gu Maojian , Wang Weigwang , Song Woo Keun , Cooper Douglas N. W. , Kaufman Stephen J. 
Published: April 2021

ABSTRACT
The α7β1 integrin was originally identified and isolated from differentiating skeletal muscle and shown to be a laminin-binding protein (Song et al. (1992)J. Cell Biol. 117, 643-657). Expression of the α7 gene and protein are developmentally regulated during skeletal muscle differentiation and have been used to identify cells at distinct stages of the myogenic lineage (George-Weinstein et al. (1993)Dev. Biol. 156, 209-229). The lactoside-binding protein L-14 exists as a dimer and has been localized on a variety of cells, in association with extracellular matrix. During myogenesis in vitro, L-14 is synthesized within replicating myoblasts but it is not secreted until these cells commence terminal differentiation and fusion into multinucleate fibers (Cooper and Barondes, J. Cell Biol. (1990) 110, 1681-1691). Addition of purified L-14 to myogenic cells plated on laminin inhibits myoblast spreading and fusion, suggesting that the L-14 lectin regulates muscle cell interactions with the extracellular matrix that are germane to myogenic development (Cooper et al. (1991)J. Cell Biol. 115, 1437-1448). We demonstrate here, using affinity chromatography and immunoblots, that α7β1 also binds to fibronectin and to the L-14 lectin. L-14 binds to both laminin and to the α7β1 integrin, and it can effectively inhibit the association of laminin and this integrin. Modulation of α7β1 interaction with its ligands by L-14 is selective: L-14 does not bind to fibronectin, nor does it interfere with the binding of fibronectin to α7β1. These results are discussed in the context of the potential roles of α7β1 in its interaction with laminin and fibronectin during myogenesis.
α-melanocyte stimulating hormone and its analogue nle4dphe7α-MSH affect morphology, tyrosinase activity and melanogenesis in cultured human melanocytes
Authors: Hunt Gillian , Todd Carole , Cresswell Janet E. , Thody Anthony J. 
Published: April 2021

ABSTRACT
Although melanocyte stimulating hormone (MSH) peptides are known to stimulate pigmentation in man, previous reports suggest that human melanocytes are relatively unresponsive to these peptides in vitro. This may be related to the conditions under which the melanocytes were cultured. Thus, we have re-investigated the in vitro effects of MSH peptides using human melanocytes cultured in the absence of artificial mitogens. Human melanocytes were incubated with α-MSH or its potent analogue Nle4DPhe7α-MSH for 3 days. After 18 hours, melanocyte morphology had evolved from mainly bipolar to dendritic in approxi-mately 66% of cultures. Nle4DPhe7α-MSH produced dose-related increases in both tyrosinase activity and melanin content although the degree of response was variable and tyrosinase activity was the relatively more responsive to the peptide. Similar results were obtained with α-MSH, but, although the effect on melanin content was similar to that of Nle4DPhe7α-MSH, the effect on tyrosinase activity was less marked. The preliminary EC50 values for the actions of the MSH peptides suggest that they may be equipotent in their actions on human melanocytes. In addition, we have demonstrated that the common melanocyte mitogens 12-O-tetradecanoyl phorbol-13-acetate (TPA) and cholera toxin affect basal melanogenesis and modulate the effects of the MSH peptides. However, not all melanocyte cultures showed melanogenic responses to the MSH peptides. Ability to respond was unrelated to basal levels of tyrosi-nase activity or melanin content. In at least some cultures, morphological and melanogenic responses appear to be independent of one another. For reasons that are unknown, a small number of human melanocyte cultures show neither morphological nor melanogenic responses to the MSH peptides. Neither peptide had any effect on cell numbers, regardless of whether the cultures were able to respond morphologically or melanogenically. The present findings are particularly interesting in light of recent devel-opments in the molecular biology of the MSH receptor.
Up-regulation of the connexin43+ GAP junction network in haemopoietic tissue before the growth of stem cells
Authors: Rosendaal Martin , Green Colin R. , Rahman Asaur , Morgan Deborah 
Published: April 2021

ABSTRACT
The early developmental stages of haemopoiesis are thought to be regulated by paracrine growth factors and by the haemopoietic environment. Are gap junctions involved here? Gap junctions are structures in cell membranes allowing the direct transfer of ions and small molecules between adjacent cells and are known to be involved in development. We have found that although connexin43 gap junctions are rare (0.00016±0.0002/μm2 tissue) in normal adult mouse marrow their expression is 80-fold higher (0.0292±0.0147/μm2) in neonatal marrow. One difference between neonatal and adult haemopoietic tissue is that in the latter more haemopoietic cells are dividing. To test if more gap junctions were due to increased division we altered adult blood-formation by mobilizing or destroying end cells - granulocytes and red cells - or by forcing stem cells to divide by making them regenerate an ablated blood-forming system. Mobilizing end cells had no effect on the number or distribution of gap junctions in marrow but forced stem cell division caused a 100-fold increase in gap junction expression and did so before any recognizable haemopoietic cells formed. There were greater than normal numbers of gap junctions in radio-protected adult mouse marrow. The cells coupled by gap junctions are TE-7+ mesodermally derived fibroblasts, STRO-1+ stromal cells, and CD45+ and CD34+ haemopoi-etic cells. We propose that there is a latent network of C×43+ gap junctions in normal quiescent marrow. In response to events that call for active division of stem cells this network is amplified and coupled to haemopoietic stem cells, perhaps enabling them to divide.
Unorthodox male meiosis in Trichosia pubescens (sciaridae)chromosome elimination involves polar organelle degeneration and monocentric spindles in first and second division
Authors: Fuge Harald 
Published: April 2021

ABSTRACT
Male meiosis in Trichosia pubescens (Sciaridae) was investigated by means of serial section electron microscopy and immunofluorescence light microscopy. From earlier studies of another sciarid fly, Sciara coprophila (Phillips (1967)J. Cell. Biol. 33, 73-92), it is known that the spindle poles in sciarid spermatogonia are characterized by pairs of ‘giant centrioles’, ring-shaped organelles composed of large numbers of singlet microtubules. In the present study sper-matocytes in early prophase of Trichosia were found to possess single giant centrioles at opposite sides of the nucleus. The obvious reduction in centriole number from the spermatogonial to the spermatocyte stage is suggested to be the result of a suppression of daughter centriole formation. In late prophase, a large aster is developed around the centriole at one pole. At the opposite pole no comparable aster is formed. Instead, a number of irregular centriolar components appear in this region, a process that is understood to be a degeneration of the polar organelle. The components of the degenerate pole migrate into a cyto-plasmic protrusion (‘bud’), which later is also utilized for the elimination of paternal chromosomes. The existence of only one functional polar centre is the reason for the formation of a monopolar monocentric spindle in first meiotic division, which in turn is one of the prerequisites for the elimination of paternal chromosomes. While the set of maternal and L chromosomes orientates and probably moves towards the pole, paternal chromosomes seem to be unable to contact the pole, possibly due to an inactivation of their kinetochores. Retrograde (‘away from the pole’) chromosome motion not involving kinetochores is assumed. Eventually, paternal chromosomes move into the pole-distal bud and are eliminated by casting off, together with the components of the degenerate polar organelle. Chro-mosome elimination can be delayed until the second meiotic division. The spindle of the second meiotic division is bipolar and monocentric. One spindle pole is marked by the polar centre of first division. The opposite spindle apex is devoid of a polar centre. It is assumed that spindle bipo-larity in the second division is induced by the amphi-ori-entated chromosomes themselves. The maternal and L chromosome set (except the non-disjunctional X chromo-some, which is found near the polar centre) congress in a metaphase plate, divide and segregate. Of the two daughter nuclei resulting from the second meiotic division, the one containing the X chromatids is retained as the nucleus of the future spermatozoon. The other nucleus becomes again eliminated within a second cytoplasmic bud.
Kinesin and tau bind to distinct sites on microtubules
Authors: Marya Pankaj K. , Syed Zarrin , Fraylich Paul E. , Eagles Peter A. M. 
Published: April 2021

ABSTRACT
We have used a fluorescent derivative of kinesin, AF-kinesin (kinesin conjugated with 5-(iodoacetamido) fluores-cein), to investigate the binding site of kinesin on micro-tubules and to compare this site with that to which tau binds. Microtubules saturated with tau will bind AF-kinesin in the presence of the ATP analogue, 5′-[β,γ-imino]triphosphate (AdoPP[NH]P). This shows that there are distinct binding sites for the two proteins. Further evidence comes from digestion studies where taxol-sta-bilised microtubules were treated with subtilisin, resulting in the cleavage of C-terminal residues from both the α-and β-tubulin subunits. These treated microtubules can no longer bind tau, but are able to bind AF-kinesin in the presence of AdoPP[NH]P. Finally, AF-kinesin will support the gliding of subtilisin-digested microtubules in the presence of ATP at rates comparable to those obtained with non-digested microtubules. These results show directly that the binding site for kinesin is outside the C-terminal region of tubulin that is removed by subtilisin and is distinct from the binding site of tau.
Comparison of normal and tumorigenic endothelial cells: Differences in thrombospondin production and responses to transforming growth factor-beta
Authors: RayChaudhury Amlan , Frazier William A. , D’Amore Patricia A. 
Published: April 2021

ABSTRACT
Cultured endothelial cells constitutively synthesize significant levels of thrombospondin, an extracellular matrix-associated protein with reported anti-angiogenic proper-ties. However, two murine endothelial cell lines, bEND.3 and Py-4-1, which have been immortalized with polyoma T oncogenes and which generate vascular malformations in vivo, produce little or no thrombospondin though bEND.3 (but not Py-4-1) growth is inhibited by the addition of exogenous thrombospondin. In addition, Py-4-1 cells are not growth-inhibited by transforming growth factor-beta, a potent endothelial inhibitor. These results indicate that these two cell lines may be useful tools in understanding the role and mechanism of action of thrombospondin and transforming growth factor-beta in endothelial cell biology. A role for thrombospondin in vascular development is further suggested by the observation of significant differ-ences in the levels of thrombospondin mRNA and protein between capillary and aortic endothelial cells. Transform-ing growth factor-beta-1 treatment of normal endothelial cells increases steady-state levels of thrombospondin mRNA and protein and results in extensive deposition of thrombospondin into the extracellular matrix. In contrast, transforming growth factor-beta-1 has little effect on thrombospondin levels in the tumorigenic endothelial cell lines. In view of our earlier finding that contact between endothelial cells and mural cells generates activated trans-forming growth factor-beta-1, and the fact that throm-bospondin is present in a fibrillar network around vascular structures in vitro, we speculate that modulation of throm-bospondin production and distribution by transforming growth factor-beta may be a physiological process to enjoin stabilization of vessels and cessation of vessel growth.
Gpi membrane anchor is determinant in intracellular accumulation of apical plasma membrane proteins in the non-polarized human colon cancer cell line HT-29 18
Authors: Nicolas F. , Tiveron M.-C. , Davoust J. , Reggio H. 
Published: April 2021

ABSTRACT
We have compared the intracellular localization of plasma membrane proteins anchored either with a transmembrane segment or with a glycosylphosphatidylinositol moiety to estimate the effects of membrane anchor on protein segregation in the non-polarized form of the human colon cancer cell line HT-29 18. We have monitored two endogenous proteins: the carcinoembryonic antigen, a glycosylphos-phatidylinositol protein and the transmembrane protein dipeptidyl peptidase IV, and two transfected proteins: the glycosylphosphatidylinositol protein Thy-1 and an engineered transmembrane form of Thy-1. Using immunocytochemistry on ultra-thin cryosections and confocal microscopy, we detected a carcinoembryonic antigen-rich vesicular compartment, excluding classical pre-lysosomal and lysosomal markers such as mannose 6-phosphate receptor, lamp-1 and cathepsin D. This compartment, where carcinoembryonic antigen accumulated, excluded the transmembrane protein dipeptidyl peptidase IV and was reduced during the polarization of the cells. Moreover, the glycosylphosphatidylinositol form of Thy-1 also accumulated in the carcinoembryonic antigen-rich compartment whereas the transmembrane form of Thy-1 was excluded. We proposed that, in the non-polarized HT-29 18 cells, accumulation of glycosylphosphatidylinositol proteins independently of transmembrane proteins reveals different intracellular fates for proteins according to their anchor in the plasma membrane.
Cell cycle regulation of the p34cdc2/p33cdk2-activating kinase p40MO15
Authors: Poon Randy Y. C. , Yamashita Katsumi , Howell Michael , Ershler Maxim A. , Belyavsky Alexander , Hunt Tim 
Published: April 2021

ABSTRACT
A key component of Cdc2/Cdk2-activating kinase (CAK) is p40MO15, a protein kinase subunit that phosphorylates the T161/T160 residues of p34cdc2/p33cdk2. The level and activity of p40MO15 were essentially constant during cleavage of fertilised Xenopus eggs and in growing mouse 3T3 cells, but serum starvation of these cells reduced both the level and activity of p40MO15. Although the level and activity of endogenous p40MO15 did not vary in the cell cycle, we found that bacterially expressed p40MO15 was activated more rapidly by M-phase cell extracts than by interphase cell extracts. Bacterially expressed p40MO15 was phosphorylated mainly on serine 170 (a p34cdc2 phosphorylation site) by mitotic cell extracts, but mutation of S170 to alanine did not affect the activation of p40MO15, whereas mutation of T176 (the equivalent site to T161/T160 in p34cdc2/p33cdk2) abolished the activation of p40MO15. These studies suggest that the level and activity of p40MO15 is probably not a major determinant of p34cdc2/p33cdk2 activity in the cell cycle, and that the activation of p40MO15 may require phosphorylation on T176.
A Rab4-like GTPase in Dictyostelium discoideum colocalizes with V-H+-ATPases in reticular membranes of the contractile vacuole complex and in lysosomes
Authors: Bush John , Nolta Kathleen , Rodriguez-Paris Juan , Kaufmann Nancy , O’Halloran Theresa , Ruscetti Tracy , Temesvari Lesly , Steck Theodore , Cardelli James 
Published: April 2021

ABSTRACT
In the course of screening a cDNA library for ras-related Dictyostelium discoideum genes, we cloned a 0.7 kb cDNA (rabD) encoding a putative protein that was 70% identical at the amino acid level to human Rab4. Rab4 is a small Mr GTPase, which belongs to the Ras superfamily and functions to regulate endocytosis in mammalian cells. Southern blot analysis indicated that the rabD cDNA was encoded by a single copy gene while Northern blot analysis revealed that the rabD gene was expressed at relatively constant levels during growth and differentiation. Affinity-purified antibodies were prepared against a RabD fusion protein expressed in bacteria; the antibodies recognized a single 23 kDa polypeptide on western blots of cell extracts. Density gradient fractionation revealed that the RabD antigen co-distributed primarily with buoyant membranes rich in vacuolar proton pumps (V-H+-ATPases) and, to a lesser extent, with lysosomes. This result was confirmed by examining cell lines expressing an epitope-tagged version of RabD. Magnetically purified early endocytic vesicles and post-lysosomal vacuoles reacted more weakly with anti-RabD antibodies than did lysosomes. Other organelles were negative for RabD. Double-label indirect immunofluorescence microscopy revealed that RabD and the 100 kDa V-H+-ATPase subunit colocalized in a fine reticular network throughout the cytoplasm. This network was reminiscent of spongiomes, the tubular elements of the contractile vacuole system. Immunoelectron microscopy confirmed the presence of RabD in lysosome fractions and in the membranes rich in V-H+-ATPase. We conclude that a Rab4-like GTPase in D. discoideum is principally associated with the spongiomes of contractile vacuole complex.
Internal structure of the 30 nm chromatin fiber
Authors: Bartolomé Salvador , Bermúdez Antonio , Daban Joan-Ramon 
Published: April 2021

ABSTRACT
In the presence of 1.7 mM Mg2+, the diameter of the circular structures produced by small chromatin fragments isolated from chicken erythrocytes remains essentially unchanged when the number of nucleosomes in these fragments increases from 10 to 36. In contrast, the results obtained in unidirectional shadowing experiments show that under the same conditions the height of the chromatin fragments increases with the number of nucleosomes. These observations indicate that the electron microscope images studied in this work correspond to a top view of small chromatin fragments. Rotary-shadowed chromatin fragments show three parts: (a) a contour with a heavy deposition of platinum; (b) an annular zone between the central region and the periphery; and (c) a central hole. The heterogeneous ring generated by the deposition of platinum in the periphery suggests that nucleosomes form a one-start helix (5–7 nucleosomes per turn) that apparently can be left- or right-handed. The annular region (thickness of about 11 nm) shows spokes probably due to flat faces and core DNA of radially oriented nucleosomes. The central hole (8-12 nm) is clearly seen in many images but it is not empty because some deformed fragments show coated material (probably linker DNA) that protrudes from this central depression. We have observed that these structural elements directly detected in short chromatin fragments are also present in long chromatin fibers. This allows us to conclude that these elements are basic structural components of the 30 nm chromatin fiber.