bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunolocalization of microfibril and microfibril-associated proteins in the subendothelial matrix of the developing mouse aorta
Authors: Davis Elaine C. 
Published: April 2021

ABSTRACT
In the developing aorta, endothelial cell connecting filaments extend from the abluminal surface of the endothelial cell to the subjacent elastic lamina. The connecting filaments are in alignment with intracellular stress fibers and are oriented parallel to the direction of blood flow. In the present study, the composition of the endothelial cell connecting filaments was investigated by indirect immuno-gold labeling with antibodies to the microfibril proteins, MP340 (fibrillin) and MAGP, and to fibronectin and heparan sulfate proteoglycan (HSPG). In the subendothelial matrix of both 15-day gestational and 5-day post-natal mouse aortae, the connecting filaments showed moderate immunoreactivity with anti-MP340; however, no significant immunoreaction was seen with anti-MAGP. Antifibronectin strongly labeled the connecting filaments and a weak immunoreaction was seen with anti-HSPG. In contrast, the adjacent ‘elastin-associated microfibrils’ showed a very strong immunoreaction with anti-MP340 and a moderate reaction with anti-MAGP. Little or no reaction was seen with anti-fibronectin or anti-HSPG. The filaments that connect endothelial cells to the subjacent elastic lamina during aortic development are thus microfibrillar in nature and related to elastin-associated microfibrils as evidenced by their positive immunoreaction with anti-MP340. The absence of labeling with anti-MAGP, however, suggests that either these fibrillin-containing filaments do not contain MAGP or that the immunoreactive epitopes are blocked by the proteins that coat the connecting filaments such as fibronectin. These results suggest that microfibrils not in association with elastin may play a role in cell anchorage and, more specifically, in the aorta may be involved in maintaining the structural integrity of the endothelial cell layer during early development of the vessel wall. Furthermore, the absence of immunoreactivity with anti-MAGP on the fibrillin-containing endothelial cell connecting filaments raises the possibility that microfibrils may consist of a family of related filaments rather than a single structural entity.
Ductus arteriosus smooth muscle cell migration on collagen: dependence on laminin and its receptors
Authors: Clyman Ronald I. , Tannenbaum Jamie , Chen Yao Qi , Cooper Douglas , Yurchenco Peter D. , Kramer Randall H. , Waleh Nahid S. 
Published: April 2021

ABSTRACT
During permanent closure of the ductus arteriosus, smooth muscle cells migrate through the extracellular matrix (ECM) to form intimal mounds that occlude the vessel’s lumen. Smooth muscle cells (SMC) migrate over surfaces coated with collagen in vitro. During the migration SMC also synthesize fibronectin (FN) and laminin (LN). Anti-bodies against FN and LN inhibit migration on collagen by 30% and 67%, respectively. Because of the apparent importance of LN in migration, we examined how SMC interact with LN and LN fragments (P1, E8, P1′, E1′, E3, E4, and G). Ductus SMC adhere to high concentrations of LN and to two fragments of the molecule: P1 and E8. They use a unique set of integrin receptors to bind to LN (α1β1, α6β1 and αVβ3), to P1 (α1β1, αVβ3), and to E8 (α6β1, αVβ3).
The αVβ3 integrin binds to the P1 fragment of LN in an RGD peptide-dependent manner, and to the E8 fragment in an RGD-independent manner; the RGD site on the P1 fragment probably is not available to the cell in intact LN. Antibodies against β1 integrins completely inhibit SMC adhesion to LN; antibodies against the αVβ3 integrin do not block SMC adhesion to LN, but do prevent cell spreading. LN is also capable of interfering with SMC adhesion to other ECM components. The antiadhesive effect of LN is located in the E1′ domain. Both exogenous and endogenous LN increase SMC motility on collagen I. The locomotion-promoting activity of LN resides in the E1′ antiadhesive domain, and not in its adhesive (P1, E8) domains. LN causes a decrease in the number of focal contacts on collagen I. This might enable SMC to alter their mobility as they move through the extracellular matrix to occlude the ductus arteriosus lumen.
Molecular cloning and characterization of a novel liver-specific transport protein
Authors: Simonson Gregg D. , Vincent Alexandra C. , Roberg Kevin J. , Huang Yue , Iwanij Victoria 
Published: April 2021

ABSTRACT
Monoclonal antibodies that specifically recognize a membrane component located on the sinusoidal domain of the hepatocyte have been used to screen a rat liver cDNA expression library and a clone encoding a novel transporter (NLT) protein has been identified. Analysis of the deduced 535 amino acid protein sequence indicates that it is unique, but shares the twelve-transmembrane domain hydrophathicity profile as well as the presence of transporter-specific amino acid motifs with bacterial and mammalian transporters. Since overall homology of NLT to known transporter genes is low (20-25% identity) it may represent a new subgroup within the transporter family of proteins. The NLT was characterized further with respect to its tissue distribution and its expression during liver development. A 2.1 kb transcript has been found in liver and at lower levels in kidney, but not in several other tissues tested. Studies on the developing liver indicate that NLT transcripts are present at a very low level from 19 through 21 gestation days with a 4- to 5-fold increase within two weeks after birth. Overall, we have cloned a novel transporter that is preferentially expressed in liver, is located on the sinusoidal domain of the plasma membrane and represents a marker for the late stage of liver development.
Surface morphology and mechanical properties of MDCK monolayers by atomic force microscopy
Authors: Hoh Jan H. , Schoenenberger Cora-Ann 
Published: April 2021

ABSTRACT
We describe the morphology and mechanical stability of the apical surface of MDCK monolayers by atomic force microscopy (AFM). Living cells could be imaged in physiological solution for several hours without noticeable deterioration. Cell boundaries appear as ridges that clearly demarcate neighboring cells. In some cases the nucleus of individual cells could be seen, though apparently only in very thin areas of the monolayer. Two types of protrusions on the surface could be visualized. Smooth bulges that varied in width from a few hundred nanometers to several micrometers, which appear to represent relatively rigid subapical structures. Another type of protrusion extended well above the membrane and was swept back and forth during the imaging. However, the microvilli that are typically present on the apical surface could not be resolved. For comparison, a transformed MDCK cell line expressing the K-rasoncogene was also examined. When cultured on solid substrata at low density, the R5 cells spread out and are less than 100 nm thick over large areas with both extensive processes and rounded edges. Many intracellular structures such as the nucleus, cytoskeletal elements and vesicles could be visualized. None of the intracellular structures seen in the AFM images could be seen by scanning electron microscopy. Both R5 cells and MDCK monolayers required imaging forces of &gt;2 nN for good image contrast. Force measurements on the MDCK monolayers show that they are very soft, with an effective spring constant of ∼0.002 N/m for the apical plasma membrane, over the first micrometer of deformation, resulting in a height deformation of approximately 500 nm per nanoNewton of applied force. The mechanical properties of the cells could be manipulated by addition of glutaraldehyde. These changes were monitored in real time by collecting force curves during the fixation reaction. The curves show a stiffening of the apical plasma membrane that was completed in ∼1 minute. On the basis of these measurements and the imaging forces required, we conclude that deformation of the plasma membrane is an important component of the contrast mechanism, in effect ‘staining’ structures based on their relative rigidity.
Receptor-mediated endocytosis is sensitive to antibodies against the uncoating ATPase (hsc70)
Authors: Höning Stefan , Kreimer Georg , Robenek Horst , Jockusch Brigitte M. 
Published: April 2021

ABSTRACT
We have investigated the functional role of the coated vesicle-uncoating ATPase (UA), a cognate heat shock protein (hsc70), in receptor-mediated endocytosis. A monoclonal antibody against bovine brain UA/hsc70 was generated that recognizes a 26 kDa proteolytic fragment harbouring the putative clathrin-binding site. In vitro, this antibody blocked the UA/hsc70-mediated release of clathrin from isolated coated vesicles (CVs). Upon microinjection into tissue culture cells, it specifically inhibited the heat shock-induced nuclear migration of UA/hsc70.
This antibody also interfered with endocytosis of ligand-receptor complexes in injected cells. Two different systems were studied: the uptake of aggregated human IgG by BHK cells transfected with a human Fc receptor (FcRII), and the internalization of LDL by human fibroblasts. Injection of the monoclonal antibody in concentrations yielding approximately equal molar ratios of antibody to enzyme resulted in a reduction of endocytosis to 20-30% of control values, as seen by conventional light and confocal laser scanning microscopy, and by electron microscopy. In the transfected BHK cells, the endocytosed ligand remained associated with the labeling for clathrin and was not delivered to the endosomal compartment within the period expected from control serum- or non-injected cells. Thin sections revealed an accumulation of coated structures in the antibody-injected cells as compared to controls. Thus, our data show that UA is essential for normal receptor-mediated endocytosis, and is presumably involved in the uncoating of CVs preceding their fusion with endosomes.
The kinetics of H1 histone kinase activation during the cell cycle of wild-type and wee mutants of the fission yeast Schizosaccharomyces pombe
Authors: Creanor J. , Mitchison J. M. 
Published: April 2021

ABSTRACT
H1 histone kinase activity has been followed in selection-synchronised cultures of fission yeast wild-type and wee1mutant cells, and in induction-synchronised cells of the mutant cdc2-33. The main conclusions are: (1) in all three cases, the peak of activity is near mitosis. (2) The rise in activity is relatively slow starting in wild type at 0.4 of the cycle before mitosis. It is proposed that the beginning of the rise is the first identifiedevent in the mitotic control. (3) The rise is twice as fast in weeand starts nearer to mitosis. (4) In all cases the beginning of the rise is in G2. (5) The fall in activity is also slow, lasting for 0.25 of the cycle, in wild type. Exit from mitosis happens well before activity has fallen to baseline. (6) In a range of size mutants, activity is roughly proportional to cell size. It is suggested that the kinase may have a cytoplasmic function. (7) Estimates have been made of the timing of mitosis in the mutants. In wee, mitosis is 0.14 of the cycle earlier than in wild type because the cells have a longer septated period at the end of the cycle. (8) A novel method has been developed for eliminating the effects of the partial asynchrony in synchronous cultures, without which the kinetic analysis would have been inaccurate.
Differential regulation of C-CAM isoforms in epithelial cells
Authors: Hunter Irene , Lindh Monica , Öbrink Björn 
Published: April 2021

ABSTRACT
C-CAM is a Ca2+-independent cell adhesion molecule (CAM) that mediates intercellular adhesion of isolated rat hepatocytes. It is widely distributed in epithelia, where its presence both at lateral cell borders and on apical cell surfaces suggests that it may have diverse biological functions. Two major isoforms, C-CAM1 and C-CAM2, which differ in the lengths of their cytoplasmic domains, have been identified. The lack of suitable in vitro systems has so far prevented a detailed study of the physiological role of C-CAM in epithelia. We now report on the identification, biochemical characterization and functional analysis of C-CAM isoforms in the established epithelial cell line NBT II, derived from a chemically induced carcinoma of rat bladder. C-CAM in NBT II cells is a 110-115 kDa cell surface glycoprotein located predominantly at sites of cell-cell contact but also present on the apical cell surface. Northern blotting analysis revealed the presence of both C-CAM1 and C-CAM2, with the major transcripts for both isoforms present within the 4.0 kb size range. The dissociation of NBT II cell colonies by anti-C-CAM antibodies indicated that at least one function of C-CAM in these cells is to mediate intercellular adhesion. The maintenance of extensive cell-cell contacts and the expression of C-CAM at the contact sites in cells grown in low Ca2+medium suggested that, like its counterpart in hepatocytes, C-CAM in NBT II cells may be a Ca2+-independent cell-cell adhesion molecule. The co-localization and coordinate reorganization of both C-CAM and actin by anti-C-CAM antibodies indicated that these two proteins were associated and suggested that interactions with the cytoskeleton may be important for the regulation of C-CAM function. The specific upregulation of C-CAM1 in cells induced to undergo epithelial to mesenchymal-like transitions (EMT) by the serum substitute Ultroser G suggested that C-CAM isoforms are important modulators of the adhesive properties of these cells.
Modulation of scatter factor/hepatocyte growth factor activity by cell-substratum adhesion
Authors: Clark Peter 
Published: April 2021

ABSTRACT
Scatter factor/hepatocyte growth factor (SF/HGF) is a multifunctional growth and motility factor whose activities vary with cell type. Here, the composition of the substratum was found to profoundly alter the scattering activities of SF/HGF, but not its mitogenetic effects, in MDCK cells. Whereas enhancement of DNA synthesis and induction of cell flattening by SF/HGF were independent of substratum composition (i.e. occurred on both fibronectin and vitronectin surfaces), colony dispersion as a result of cell separation fails to occur or is markedly reduced on surfaces where vitronectin is the major adhesive ligand. Prolonged exposure of non-scattering cultures to SF/HGF resulted in cells at colony margins producing long protrusions, which indicate that the motility of these cells is stimulated but ‘frustrated’ by the lack of breakdown of cell-cell adhesion. Scattering therefore appears to comprise two major components: increased motility and breakdown of cell-cell adhesion. The pathway leading to the breakdown of cell-cell contacts is modulated by downstream signals from extracellular matrix receptors. When cultured on immobilised fibronectin, vitronectin or a surface containing both, colony dissociation correlates with the presence of fibronectin, suggesting that positive signals from fibronectin receptors are required for SF/HGF-induced cell separation. Comparison of the findings in this study with those of a recent report on the modulation of SF/HGF-induced tubulogenesis by ECM (Santos, O. F. P. and Nigam, S. K. (1993) Dev. Biol. 160, 293-302), where vitronectin in type-1 collagen gels alters the pattern of SF/HGF-induced MDCK tubule formation from highly branched to long and unbranched, suggests that cell motility enhancement leads to tubule formation whereas the breakdown of cell-cell adhesion is required for tubule branching.
Nuclear mitotic apparatus protein (NuMA): spindle association, nuclear targeting and differential subcellular localization of various numa isoforms
Authors: Tang Tang K. , Tang Chieh-ju C. , Chao Yu-Jane , Wu Cheng-Wen 
Published: April 2021

ABSTRACT
We have recently shown that the nuclear mitotic apparatus protein (NuMA) is composed of at least three isoforms that differ mainly at the carboxy terminus, and are generated by alternative splicing of a common mRNA precursor from a single NuMA gene (J. Cell Sci. (1993) 104, 249-260). Transient expression of human NuMA-l isoform (T33/p230) in Chinese hamster ovary polyoma (CHOP) cells showed that NuMA-l was present in interphase nuclei and was concentrated at the polar regions of the spindle apparatus in mitotic cells. However, expression of two other isoforms (NuMA-m and -s) revealed a distinct sub-cellular localization. NuMA-m (U4/p195) and NuMA-s (U6/p194) were present in the interphase cytosol and appeared to be mainly located at the centrosomal region. When cells entered into mitosis, however, NuMA-m and −s moved to the mitotic spindle pole. Analysis of a series of linker scanning-mutants and NuMA/β-galactosidase chimeric proteins showed that residues 1972-2007 of NuMA-l constitute a novel nuclear localization signal (NLS) and residues 1538-2115 are necessary and sufficient for spindle association. Further analysis of the NLS by site-specific mutagenesis indicated that Lys1988 is essential for nuclear targeting, whereas Arg1984 is not. These results have allowed us tentatively to assign specific biological activities to distinct structural domains of the NuMA polypeptide.
A monoclonal antibody identifies a novel GPI-anchored glycoprotein involved in epithelial intercellular adhesion
Authors: Rabino Mauro , Trusolino Livio , Prat Maria , Cremona Ottavio , Savoia Paola , Marchisio Pier Carlo 
Published: April 2021

ABSTRACT
BD31 mAb, raised against a line of gastric carcinoma cells, reacts with intercellular boundaries of human transformed cells originating from carcinomas or sarcomas growing in epithelial-like clusters as well as in primary cultures of epithelial and endothelial cells. BD31 also reacts with inter- cellular rims of normal and transformed epithelial tissues and is particularly abundant in glands and fast-growing epithelia but absent in nervous and muscle tissues as well as in blood and in mesenchyme-derived cells. Confocal analysis indicates that BD31 is located in dots at cell-cell contacts but not in basal and apical domains of cultured and in situ epithelial cells. mAb BD31 precipitates a 100 kDa protein from cells labeled with [35S]methionine or [3H]glucosamine as well as from 125I-surface-labeled cells. This glycoprotein resists to trypsin in the presence of Ca2+, releases an 80 kDa fragment in the medium and does not react with antibodies to the conserved cytodomain of known cadherins or, specifically, to the ectodomain of E- cadherin in western blotting; moreover, the lack of cadherin cytodomain and protein removal by phospho- inositide-specific phospholipase indicate its membrane anchoring by a glycosyl-phosphatidylinositol (GPI) moiety. BD31 IgGs do not impair cell-matrix adhesion but induce inhibition of Ca2+-dependent aggregation, loss of cell-cell adhesion, scattering of confluent cells and appearance of migratory cell phenotypes in a dose- and time-dependent manner. This novel GPI-anchored glycoprotein may regulate intercellular adhesion by a mechanism involving membrane-associated phospholipases.
Cell cycle analysis and chromosomal localization of human Plk1, a putative homologue of the mitotic kinases Drosophila polo and Saccharomyces cerevisiae Cdc5
Authors: Golsteyn Roy M. , Schultz Sharon J. , Bartek Jiri , Ziemiecki Andrew , Ried Thomas , Nigg Erich A. 
Published: April 2021

ABSTRACT
Polo and CDC5 are two genes required for passage through mitosis in Drosophila melanogaster and Saccharomyces cerevisiae, respectively. Both genes encode structurally related protein kinases that have been implicated in regu- lating the function of the mitotic spindle. Here, we report the characterization of a human protein kinase that displays extensive sequence similarity to Drosophila polo and S. cerevisiae Cdc5; we refer to this kinase as Plk1 (for polo-like kinase 1). The largest open reading frame of the Plk1 cDNA encodes a protein of 68,254 daltons, and a protein of this size is detected by immunoblotting of HeLa cell extracts with monoclonal antibodies raised against the C-terminal part of Plk1 expressed in Escherichia coli. Northern blot analysis of RNA isolated from human cells and mouse tissues shows that a single Plk1 mRNA of 2.3 kb is highly expressed in tissues with a high mitotic index, con- sistent with a possible function of Plk1 in cell proliferation. The Plk1 gene maps to position p12 on chromosome 16, a locus for which no associations with neoplastic malignan- cies are known. The Plk1 protein levels and its distribution change during the cell cycle, in a manner consistent with a role of Plk1 in mitosis. Thus, like Drosophila polo and S. cerevisiae Cdc5, human Plk1 is likely to function in cell cycle progression.
A cytoplasmic dynein motor in Drosophila: identification and localization during embryogenesis
Authors: Hays Tom S. , Porter Mary E. , McGrail Maura , Grissom Paula , Gosch Pam , Fuller Margaret T. , McIntosh J. Richard 
Published: April 2021

ABSTRACT
We have characterized a cytoplasmic dynein motor isoform that is present in extracts of Drosophila embryos. A prominent high molecular weight (HMW) polypeptide (&gt;400 kDa) is enriched in microtubules prepared from nucleotide-depleted embryonic extracts. Based on its ATP-sensitive microtubule binding activity, 20 S sedimentation coefficient, sensitivity to UV-vanadate and nucleotide specificity, the HMW polypeptide resembles cytoplasmic dyneins prepared from other organisms. The Drosophila cytoplasmic dynein acts as a minus-end motor that promotes microtubule translocation in vitro. A polyclonal antibody raised against the dynein heavy chain polypeptide was used to localize the dynein antigen in whole-mount preparations of embryos by immunofluorescence microscopy. These studies show that the dynein motor is associated with microtubules throughout embryogenesis, including mitotic spindle microtubules and microtubules of the embryonic nervous system.
The cytoplasmic domain of syndecan-1 is not required for association with Triton X-100-insoluble material
Authors: Miettinen Heini M. , Jalkanen Markku 
Published: April 2021

ABSTRACT
Cell surface heparan sulfate proteoglycans such as syndecan-1 bind various extracellular matrix proteins and have been suggested to interact with the cytoskeleton. Such interactions are thought to be important for stabilizing cell morphology. Syndecan-1 resists extraction with Triton X-100. This insolubility was reported not to be affected by removal of the glycosaminoglycan chains, suggesting that the insolubility is not due to binding to the extracellular matrix, but rather to an association with the actin cytoskeleton (Rapraeger, A., Jalkanen, M. and Bernfield, M. (1986) J. Cell Biol. 103, 2683-2696). To examine further the interaction of syndecan-1 with the Triton X-100-insoluble residue, we expressed wild-type mouse syndecan-1 and a cytoplasmic deletion mutant (tail-less) in Chinese hamster ovary cells. We observed that both the wild-type and the tail-less syndecan-1 were partly insoluble in Triton X-100. The insolubility was not affected by increasing temperature (37°C or 50°C) or by cytochalasin D. Removal of the glycosaminoglycan chains from the ectodomain, however, resulted in complete Triton X-100 solubility, unlike previous reports. Syndecan-1 could also be released into the Triton X-100-soluble fraction by addition of heparin or heparan sulfate to the extraction medium. We conclude that the cytoplasmic domain of syndecan-1 is not responsible for Triton X-100 insolubility. Instead, our results indicate that Triton X-100 insolubility is caused by an interaction of syndecan-1 molecules with other cellular and/or extracellular molecules mediated by the heparan sulfate chains.