bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of the β6 integrin subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling
Authors: Breuss J. M. , Gallo J. , DeLisser H. M. , Klimanskaya I. V. , Folkesson H. G. , Pittet J. F. , Nishimura S. L. , Aldape K. , Landers D. V. , Carpenter W. , Gillett N. , Sheppard D. , Matthay M. A. , Albelda S. M. , Kramer R. H. , Pytela R. 
Published: April 2021

ABSTRACT
The αvβ6 integrin was identified in cultured epithelial cells and functions as a fibronectin receptor. We have now used monoclonal antibodies to determine in vivo expression patterns of the β6 subunit in normal and pathological human or primate tissues, and during experimental wound healing or induced lung injury. The results indicate that β6 expression is restricted to epithelia and is up-regulated in parallel with morphogenetic events, tumorigenesis, and epithelial repair. During development of the kidney, lung, and skin, we found that β6 is expressed by specific types of epithelial cells, whereas it is mostly undetectable in normal adult kidney, lung and skin. In contrast, we detected high-level expression in several types of carcinoma. For example, β6 is almost invariably neo-expressed in squamous cell carcinomas derived from the oral mucosa, often focally localized at the infiltrating edges of tumor islands.
Expression of β6 is also upregulated in migrating keratinocytes at the wound edge during experimental epidermal wound healing. Similarly, β6 expression is induced in type II alveolar epithelial cells during lung injury caused by injection of live bacteria. We also observed β6 expression in adult lungs and kidneys at focal sites of subclinical inflammation, as well as in a variety of clinical specimens from patients with chronic or acute inflammation of the lungs or kidneys. From these findings and earlier results, we hypothesize that αvβ6 affects cell spreading, migration and growth during reorganization of epithelia in development, tissue repair, and neoplasia.
Visualization of exocytosis during sea urchin egg fertilization using confocal microscopy
Authors: Terasaki Mark 
Published: April 2021

ABSTRACT
A Ca2+ wave at fertilization triggers cortical granule exocytosis in sea urchin eggs. New methods for visualizing exocytosis of individual cortical granules were developed using fluorescent probes and confocal microscopy. Electron microscopy previously provided evidence that cortical granule exocytosis results in the formation of long-lived depressions in the cell surface. Fluorescent dextran or ovalbumin in the sea water seemed to label these depressions and appeared by confocal microscopy as disks. FM 1-43, a water-soluble fluorescent dye which labels membranes in contact with the sea water, seemed to label the membrane of these depressions and appeared as rings. In double-labeling experiments, the disk and ring labeling by the two types of fluorescent dyes were coincident to within 0.5 second. The fluorescent labeling is coincident with the disappearance of cortical granules by transmitted light microscopy, demonstrating that the labeling corresponds to cortical granule exocytosis. Fluorescent labeling was simultaneous with an expansion of the space occupied by the cortical granule, and labeling by the fluorescent dextran was found to take 0.1-0.2 second. These results are consistent with, and reinforce the previous electron microscopic evidence for, long-lived depressions formed by exocytosis; in addition, the new methods provide new ways to investigate cortical granule exocytosis in living eggs.
The fluorescence labeling methods were used with the Ca2+ indicator Ca Green-dextran to test if Ca2+ and cortical granule exocytosis are closely related spatially and temporally. In any given region of the cortex, Ca2+ increased relatively slowly. Furthermore, Ca2+ began to rise about 8 seconds before the first fluorescent labeling of exocytosis, suggesting that it takes a relatively long time for the Ca2+ concentration to reach a threshold value that triggers fusion. No evidence was found for highly localized Ca2+ increases that could account for the pattern of individual exocytic events. These results indicate that there is a long lag period between Ca2+ rise and cortical granule exocytosis, and that Ca2+ and cortical granule exocytosis are not tightly coupled spatially and temporally.
Modulation of melanogenic protein expression during the switch from eu-to pheomelanogenesis
Authors: Kobayashi Takeshi , Vieira Wilfred D. , Potterf Brian , Sakai Chie , Imokawa Genji , Hearing Vincent J. 
Published: April 2021

ABSTRACT
Mammalian melanocytes can produce two basic types of melanin, eumelanin and pheomelanin, within discrete organelles termed melanosomes. The physiological signals that regulate this switch are extrinsic to the melanocyte, and include α-melanocyte stimulating hormone and the agouti protein. Tyrosinase, encoded at the albino locus, is the enzyme essential for the synthesis of both types of melanin, but other tyrosinase-related proteins (e.g. TRP1 encoded at the brown locus and TRP2 encoded at the slaty locus) regulate eumelanogenesis catalytically at steps distal to tyrosinase (as 5,6-dihydroxyindole-2-carboxylic acid oxidase and DOPAchrome tautomerase, respectively). The silver protein is another melanosomal protein, and although it has some limited homology to the tyrosinase-related proteins, it does not have any known enzymatic function and probably serves as a structural matrix protein. The role of each of those melanosomal proteins in pheomelanogenesis, however, is still unclear. In this study, we have compared the expression and catalytic functions of those proteins in pheomelanic and eumelanic hair bulb melanocytes. There was no detectable expression of TRP1 or TRP2, or either of their enzymatic activities, in hair bulbs of lethal yellow (Ay/a) newborn mice, and tyrosinase activity was present at a reduced level compared to that found in hair bulbs of black (a/a) newborn mice. Similar results were observed in regenerating hair bulbs of adult lethal yellow mice and in hair bulbs of 5-to 7-day-old agouti mice (A/A), an age where pheomelanin is produced predominantly. Expression of the silver protein was similarly not observed in hair bulbs of the pheomelanic mice. These results support the hypothesis that TRP1 and TRP2 may have only eumelanogenesis-specific functions and further suggest that the silver protein has a structural and/or unknown catalytic function that is similarly eumelanogenesis-specific. Expression of TRP1, TRP2 and/or the silver protein is not required for the switch to pheomelanogenesis, since their functions are specifically down-regulated by the action of the agouti protein, the physiological regulator of the switch to produce pheomelanin.
Cytoskeletal filament assembly and the control of cell spreading and function by extracellular matrix
Authors: Mooney David J. , Langer Robert , Ingber Donald E. 
Published: April 2021

ABSTRACT
This study was undertaken to analyze how cell binding to extracellular matrix produces changes in cell shape. We focused on the initial process of cell spreading that follows cell attachment to matrix and, thus, cell ‘shape’ changes are defined here in terms of alterations in projected cell areas, as determined by computerized image analysis. Cell spreading kinetics and changes in microtubule and actin microfilament mass were simultaneously quantitated in hepatocytes plated on different extracellular matrix substrata. The initial rate of cell spreading was highly dependent on the matrix coating density and decreased from 740 μm2/h to 50 μm2/h as the coating density was lowered from 1000 to 1 ng/cm2. At approximately 4 to 6 hours after plating, this initial rapid spreading rate slowed and became independent of the matrix density regardless of whether laminin, fibronectin, type I collagen or type IV collagen was used for cell attachment. Analysis of F-actin mass revealed that cell adhesion to extracellular matrix resulted in a 20-fold increase in polymerized actin within 30 minutes after plating, before any significant change in cell shape was observed. This was followed by a phase of actin microfilament disassembly which correlated with the most rapid phase of cell extension and ended at about 6 hours; F-actin mass remained relatively constant during the slow matrix-independent spreading phase. Microtubule mass increased more slowly in spreading cells, peaking at 4 hours, the time at which the transition between rapid and slow spreading rates was observed. However, inhibition of this early rise in microtubule mass using either nocodazole or cycloheximide did not prevent this transition. Use of cytochalasin D revealed that microfilament integrity was absolutely required for hepatocyte spreading whereas interference with microtubule assembly (using nocodazole or taxol) or protein synthesis (using cycloheximide) only partially suppressed cell extension. In contrast, cell spreading could be completely inhibited by combining sub-optimal doses of cytochalasin D and nocodazole, suggesting that intact microtubules can stabilize cell form when the microfilament lattice is partially compromised. The physiological relevance of the cytoskeleton and cell shape in hepatocyte physiology was highlighted by the finding that a short exposure (6 hour) of cells to nocodazole resulted in production of smaller cells 42 hours later that exhibited enhanced production of a liver-specific product (albumin). These data demonstrate that spreading and flattening of the entire cell body is not driven directly by net polymerization of either microfilaments or microtubules. Instead, extracellular matrix appears to control cell shape and function by producing global changes in a structurally integrated cytoskeletal network.
gp330 on type ii pneumocytes mediates endocytosis leading to degradation of pro-urokinase, plasminogen activator inhibitor-1 and urokinase-plasminogen activator inhibitor-1 complex
Authors: Stefansson Steingrimur , Kounnas Maria Z. , Henkin Jack , Mallampalli Rama K. , Chappell David A. , Strickland Dudley K. , Argraves W. Scott 
Published: April 2021

ABSTRACT
Glycoprotein 330 (gp330) is a member of a family of receptors related to the low density lipoprotein receptor (LDLR). Although several ligands have been shown to bind gp330 in solid-phase assays, the ability of gp330 to mediate ligand endocytosis has not been demonstrated. To develop a cellular model for gp330 function we screened a variety of cultured cell lines and identified several that expressed this protein, including immortalized rat type II pneumocytes and a human and two rodent tumor cell lines. Using type II pneumocytes, endocytosis of a previously described gp330 ligand, urokinase (uPA) complexed with plasminogen activator inhibitor-1 (uPA:PAI-1) and two new ligands, PAI-1 and pro-uPA, was demonstrated. RAP, the 39 kDa receptor-associated protein known to antagonize ligand binding to gp330 in solid-phase binding assays, completely inhibited both internalization and degradation of the radiolabeled ligands by type II pneumocytes. This suggested that the clearance of these ligands was dependent on either gp330 or the LDLR-related protein (LRP), which shares several ligand-binding characteristics with gp330. By using polyclonal antibodies to gp330, the cellular internalization and degradation of the ligands were inhibited by 30-50%; remaining ligand internalization and degradation activity could be partially inhibited by polyclonal antibodies against LRP. These findings indicate that gp330, like other LDLR family members, mediates endocytosis of its ligands. In addition, gp330 acts in concert with LRP in type II pneumocytes to mediate clearance of a variety of proteins involved in plasminogen activation, including uPA:PAI-1 complexes PAI-1 and pro-uPA. The evidence that gp330 can mediate clearance of these proteins in cultured cells implicates it as having a role in extracellular protease regulation in the many specialized epithelia where it is expressed in vivo.
Chromatin diminution is strictly correlated to somatic cell behavior in early development of the nematode Parascaris univalens
Authors: Esteban M. R. , Giovinazzo G. , Goday C. 
Published: April 2021

ABSTRACT
We have studied the relationship between the occurrence of chromatin diminution and the developmental behavior of somatic blastomeres in early development of the nematode Parascaris univalens. A cytological and immuno-cytochemical analysis of chromatin diminution was performed in P. univalens embryos exposed to ‘vegetalizing’ (LiCl) and ‘animalizing’ (NaSCN) substances during early developmental stages. We have also analyzed chromatin diminution in embryos displaying only symmetrical ‘somatic-like’ divisions due to a brief cytochalasin B treatment at the pronuclear stage. The results show that LiCl treatment induces chromatin diminution in P0-P4 pregerminal blastomeres while NaSCN treatment prevents it. Pregerminal cells undergoing chromatin diminution in ‘vegetalized’ embryos behaved like somatic cells with respect to division and cleavage patterns. NaSCN treatment results in undiminuted polynucleated embryos that are not capable of cleavage. In cytochalasin B-pulsed embryos, chromatin diminution occurs in all blastomeres. From our results we conclude that chromatin diminution and somatic cell behavior are not separable and constitute strictly correlated events in Parascaris. Moreover, the results indicate that the segregation of the cytoplasmic factors involved in chromatin diminution in early Parascaris development are microfilament-mediated. Here, we also report the formation of a latter pregerminal cell precursor (P5) not susceptible to LiCl-induced chromatin diminution.
caenorhabditis elegans cyclin a- and b-type genes: A cyclin a multigene family, an ancestral cyclin b3 and differential germline expression
Authors: Kreutzer Monique A. , Richards James P. , Silva-Udawatta Mihiri N. De , Temenak Joseph J. , Knoblich Jürgen A. , Lehner Christian F. , Bennett Karen L. 
Published: April 2021

ABSTRACT
We have cloned cDNAs for Caenorhabditis elegans cyclins A1, B and B3. While cyclins A1 and B are most closely related to either A- or B-type cyclins of other species, cyclin B3 is less related to these cyclins. However, this cyclin is most similar to the recently identified chicken cyclin B3. Our identification of a Caenorhabditis homolog demonstrates that cyclin B3 has been conserved in evolution. cyclin A1 is a member of an A-type multigene family; however the cyclin A1 cDNA only recognizes a single band on northern blots. A single-sized RNA is also observed for the cyclin B3 cDNA. In contrast, three different transcripts are observed for the cyclin B cDNA. Based on our analyses using RNAs from germline-defective mutants and from populations enriched for males, one cyclin B transcript is specific to the paternal germline. The two other cyclin B transcripts, as well as the cyclin A1 and cyclin B3 transcripts, are most abundant in the maternal germline and are only present at low levels in other tissues. Moreover, the 3′ untranslated regions of each Caenorhabditis cyclin cDNA possess several copies of potential translational control elements shown in Xenopus and Drosophila maternal cyclin mRNAs to function during oogenesis and early embryogenesis.
Invasion of Toxoplasma gondii occurs by active penetration of the host cell
Authors: Morisaki J. Hiroshi , Heuser John E. , Sibley L. David 
Published: April 2021

ABSTRACT
Toxoplasma gondii is an obligate intracellular parasite that infects a wide variety of vertebrate cells including macrophages. We have used a combination of video microscopy and fluorescence localization to examine the entry of Toxoplasma into macrophages and nonphagocytic host cells. Toxoplasma actively invaded host cells without inducing host cell membrane ruffling, actin microfilament reorganization, or tyrosine phosphorylation of host proteins. Invasion occurred rapidly and within 25-40 seconds the parasite penetrated into a tight-fitting vacuole formed by invagination of the plasma membrane. In contrast, during phagocytosis of Toxoplasma, extensive membrane ruffling captured the parasite in a loose-fitting phagosome that formed over a period of 2-4 minutes. Phagocytosis involved both reorganization of the host cytoskeleton and tyrosine phosphorylation of host proteins. In some cases, parasites that were first internalized by phagocytosis, were able to escape from the phagosome by a process analogous to invasion. These studies reveal that active penetration of the host cell by Toxoplasma is fundamentally different from phagocytosis or induced endocytic uptake. The novel ability to penetrate the host cell likely contributes to the capability of Toxoplasma-containing vacuoles to avoid endocytic processing.
The α2β1 integrin regulates collagen-mediated mdck epithelial membrane remodeling and tubule formation
Authors: Schwimmer Randi , Ojakian George K. 
Published: April 2021

ABSTRACT
Previous studies have demonstrated that incubation of MDCK cell epithelial cysts in collagen gel induced a reversal in cell surface polarity that was regulated by β1 integrins. Further experiments were done to identify the specific collagen binding integrin involved by applying collagen gel overlays to the apical membrane of subconfluent MDCK monolayers. Cell surface levels of the apical membrane glycoprotein gp135 were monitored by ELISA to quantitate the extent of collagen-mediated membrane remodeling. After an 8 hour incubation with collagen, there was a 35% reduction in gp135 while the cell surface levels of the α2, α3 and β1 integrin subunits were not affected. Immunofluorescence microscopy confirmed the loss of gp135 from selected regions of the apical cell surface while the α2 and β1 integrin subunits were distributed in small clusters over the entire apical membrane in both control and collagen-treated monolayers. Collagen-mediated loss of gp135 was inhibited by monoclonal antibodies which recognize either the α2 or β1 integrin subunits but not by a monoclonal antibody against the α6β1 integrin. These results demonstrated that remodeling of the apical membrane had occurred, allowing the selective retention of β1 integrins but not gp135. They were supported by the observation that collagen-mediated loss of apical membrane microvilli was inhibited by the monoclonal antibody against the α2 integrin subunit. Incubation of confluent monolayers with collagen gel induced the formation of polarized epithelial tubules within 16 hours. Epithelial tubule biogenesis was completely inhibited by monoclonal antibodies against either the α2 or β1 integrin subunits, providing strong evidence that the α2β1 integrin is essential for collagen-mediated epithelial membrane remodeling and tubule formation.
Mapping of nucleoporins to the center of the nuclear pore complex by post-embedding immunogold electron microscopy
Authors: Grote Monika , Kubitscheck Ulrich , Reichelt Rudolf , Peters Reiner 
Published: April 2021

ABSTRACT
Ultrathin sections of Lowicryl K4M embedded cultured 3T3 cells, human keratinocytes and mouse/rat liver tissue were incubated with polyspecific primary antibodies against p62 and other nucleoporins followed by 10 nm gold labeled secondary antibodies. By quantitatively evaluating both cross sections and tangential sections of the NPC, we found that irrespective of the cell type antibodies predominantly bound within a radius of 25 nm around the central axis of the nuclear pore complex (NPC). Superposition of a current structural model of the NPC with the nucleoporin distribution observed by us showed that nucleoporins mapped predominantly to the controversely discussed ‘central granule’. Our experimental approach was verified by mapping gp210, another nuclear pore protein, at or very close to the NPC in the perinuclear cisterna thus establishing a distribution pattern completely different from that of the nucleoporins.
Monoclonal and polyclonal antibodies detect a new type of post-translational modification of axonemal tubulin
Authors: Levilliers Nicolette , Fleury Anne , Hill Anne-Marie 
Published: April 2021

ABSTRACT
Polyclonal (PAT) and monoclonal (AXO 49) antibodies against Paramecium axonemal tubulin were used as probes to reveal tubulin heterogeneity. The location, the nature and the subcellular distribution of the epitopes recognized by these antibodies were, respectively, determined by means of: (i) immunoblotting on peptide maps of Paramecium, sea urchin and quail axonemal tubulins; (ii) immunoblotting on ciliate tubulin fusion peptides generated in E. coli to discriminate antibodies directed against sequential epitopes (reactive) from post-translational ones (non reactive); and (iii) immunofluorescence on Paramecium cells, using throughout an array of antibodies directed against tubulin sequences and post-translational modifications as references.
AXO 49 monoclonal antibody and PAT serum were both shown to recognize epitopes located near the carboxylterminal end of both subunits of Paramecium axonemal tubulin, whereas the latter recognized additional epitopes in α-tubulin; AXO 49 and a fraction of the PAT serum proved to be unreactive over fusion proteins; both PAT and AXO 49 labelled a restricted population of very stable microtubules in Paramecium, consisting of axonemal and cortical ones, and their reactivity was sequentially detected following microtubule assembly; finally, both antibodies stained two upward spread bands in Paramecium axonemal tubulin separated by SDS-PAGE, indicating the recognition of various α- and β-tubulin isoforms displaying different apparent molecular masses.
These data, taken as a whole, definitely establish that PAT and AXO 49 recognize a post-translational modification occurring in axonemal microtubules of protozoa as of metazoa. This modification appears to be distinct from the previously known ones, and all the presently available evidence indicates that it corresponds to the very recently discovered polyglycylation of Paramecium axonemal α- and β-tubulin.
Osteoclast ATP receptor activation leads to a transient decrease in intracellular pH
Authors: Yu Hesheng , Ferrier Jack 
Published: April 2021

ABSTRACT
Application of extracellular adenosine triphosphate (ATP) induces a pulsed decrease in osteoclast intracellular pH (pHi), as measured with seminaphthofluorescein (SNAFL)-calcein on a laser scanning confocal microscope. Adenosine diphosphate also produces a pHi decrease, but adenosine monophosphate, uridine triphosphate, 2-methylthio-ATP, and β,γ-methylene-ATP have little effect on pHi. The ATP-induced pHi decrease is largely inhibited by suramin, a P2 purinergic receptor blocker. Clamping intracellular free [Ca2+] ([Ca2+]i) with BAPTA/AM does not affect the ATP-induced pHi change, showing that this pHi decrease is not caused by the increased intracellular [Ca2+]i that is produced by activation of osteoclast purinergic receptors. We show that an increase in [Ca2+]i by itself will produce a pHi increase. The ATP effect is not blocked by inhibition of Na+/H+ exchange by either Na+-free bathing medium or amiloride. Two inhibitors of the osteoclast cell membrane proton pump, N-ethylmaleimide and vanadate, produce partial inhibition of the ATP-induced pHi decrease. Two other proton pump inhibitors, bafilomycin and N,N′-dicyclohexylcarbodiimide, have no influence on the ATP effect. None of the proton pump inhibitors but vanadate has a direct effect on pHi. Vanadate produces a transient pHi increase upon application to the bathing medium, possibly as a result of its known effect of stimulating the Na+/H+ exchanger. Inhibition of Cl−/HCO3− exchange by decreasing extracellular Cl− gives a pronounced long-term pHi increase, supporting the hypothesis that this exchange has an important role in osteoclast pHi homeostasis. In Cl−-free extracellular medium, there is a greatly reduced effect of extracellular ATP on pHi. The ATP effect is partially inhibited by diisothiocyanatostilbene sulfonic acid, an inhibitor of the Cl−/HCO3− exchanger. These data provide evidence that ATP binding to a P2 purinergic receptor results in a transient enhancement of Cl− /HCO3− exchange across the osteoclast cell membrane.