Studying Extracellular Signaling Utilizing a Glycoproteomic Approach: Lectin Blot Surveys, a First and Important Step

Coupling between osteoblast-mediated bone formation and osteoclast-mediated bone resorption maintains both mechanical integrity and mineral homeostasis. Zinc is required for the formation, mineralization, growth, and maintenance of bones. We examined the effects of zinc sulfate on osteoblastic differentiation of human periosteum-derived cells (hPDCs) and osteoclastic differentiation of THP-1 cells. Zinc sulfate enhanced the osteoblastic differentiation of hPDCs; however, it did not affect the osteoclastic differentiation of THP-1 cells. The levels of extracellular signaling-related kinase (ERK) were strongly increased during osteoblastic differentiation in zinc sulfate-treated hPDCs, compared with other mitogen-activated protein kinases (MAPKs). Zinc sulfate also promoted osteogenesis in hPDCs and THP-1 cells co-cultured with the ratio of one osteoclast to one osteoblast, as indicated by alkaline phosphatase levels, mineralization, and cellular calcium contents. In addition, the receptor activator of nuclear factor kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio was decreased in the zinc sulfate-treated co-cultures. Our results suggest that zinc sulfate enhances osteogenesis directly by promoting osteoblastic differentiation and osteogenic activities in osteoblasts and indirectly by inhibiting osteoclastic bone resorption through a reduced RANKL/OPG ratio in co-cultured osteoblasts and osteoclasts.

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The signaling role of extracellular ATP in co-culture of Shiraia sp. S9 and Pseudomonas fulva SB1 for enhancing hypocrellin A production

Microbial Cell Factories ◽

10.1186/s12934-021-01637-9 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Xin Ping Li ◽

Lu Lu Zhou ◽

Yan Hua Guo ◽

Jian Wen Wang

Keyword(s):

Atp Release ◽

Specific Inhibitor ◽

Extracellular Atp ◽

Oxidase Inhibitor ◽

Disulfonic Acid ◽

Early Event ◽

Fungal Metabolites ◽

Extracellular Signaling ◽

Hypocrellin A

Abstract Background Adenosine 5′-triphosphate (ATP) plays both a central role as an intracellular energy source, and a crucial extracellular signaling role in diverse physiological processes of animals and plants. However, there are less reports concerning the signaling role of microbial extracellular ATP (eATP). Hypocrellins are effective anticancer photodynamic therapy (PDT) agents from bambusicolous Shiraia fungi. The co-culture of Shiraia sp. S9 and a bacterium Pseudomonas fulva SB1 isolated from Shiraia fruiting bodies was established for enhanced hypocrellin A (HA) production. The signaling roles of eATP to mediate hypocrellin biosynthesis were investigated in the co-culture. Results The co-culture induced release of eATP at 378 nM to the medium around 4 h. The eATP release was interdependent on cytosolic Ca2+ concentration and reactive oxygen species (ROS) production, respectively. The eATP production could be suppressed by the Ca2+ chelator EGTA or abolished by the channel blocker La3+, ROS scavenger vitamin C and NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). The bacterium-induced H2O2 production was strongly inhibited by reactive blue (RB), a specific inhibitor of membrane purinoceptors, but dependent on the induced Ca2+ influx in the co-culture. On the other hand, the application of exogenous ATP (exATP) at 10–300 µM to Shiraia cultures also promoted fungal conidiation and HA production, both of which were blocked effectively by the purinoceptor inhibitors pyridoxalphosphate-6-azophenyl-2′, 4′-disulfonic acid (PPADS) and RB, and ATP hydrolase apyrase. Both the induced expression of HA biosynthetic genes and HA accumulation were inhibited significantly under the blocking of the eATP or Ca2+ signaling, and the scavenge of ROS in the co-culture. Conclusions Our results indicate that eATP release is an early event during the intimate bacterial–fungal interaction and eATP plays a signaling role in the bacterial elicitation on fungal metabolites. Ca2+ and ROS are closely linked for activation of the induced ATP release and its signal transduction. This is the first report on eATP production in the fungal–bacterial co-culture and its involvement in the induced biosynthesis of fungal metabolites. Graphic abstract

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Ras-mediated activation of the TORC2–PKB pathway is critical for chemotaxis

The Journal of Cell Biology ◽

10.1083/jcb.201001129 ◽

2010 ◽

Vol 190 (2) ◽

pp. 233-245 ◽

Cited By ~ 78

Author(s):

Huaqing Cai ◽

Satarupa Das ◽

Yoichiro Kamimura ◽

Yu Long ◽

Carole A. Parent ◽

...

Keyword(s):

Cell Migration ◽

Actin Polymerization ◽

Time Course ◽

Specific Inhibitor ◽

Ras Proteins ◽

Extracellular Signaling ◽

Upstream Regulator ◽

Pkb Phosphorylation ◽

G Protein Coupled

In chemotactic cells, G protein–coupled receptors activate Ras proteins, but it is unclear how Ras-associated pathways link extracellular signaling to cell migration. We show that, in Dictyostelium discoideum, activated forms of RasC prolong the time course of TORC2 (target of rapamycin [Tor] complex 2)-mediated activation of a myristoylated protein kinase B (PKB; PKBR1) and the phosphorylation of PKB substrates, independently of phosphatidylinositol-(3,4,5)-trisphosphate. Paralleling these changes, the kinetics of chemoattractant-induced adenylyl cyclase activation and actin polymerization are extended, pseudopodial activity is increased and mislocalized, and chemotaxis is impaired. The effects of activated RasC are suppressed by deletion of the TORC2 subunit PiaA. In vitro RasCQ62L-dependent PKB phosphorylation can be rapidly initiated by the addition of a PiaA-associated immunocomplex to membranes of TORC2-deficient cells and blocked by TOR-specific inhibitor PP242. Furthermore, TORC2 binds specifically to the activated form of RasC. These results demonstrate that RasC is an upstream regulator of TORC2 and that the TORC2–PKB signaling mediates effects of activated Ras proteins on the cytoskeleton and cell migration.

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Visible-light-initiated hydrogels preserving cartilage extracellular signaling for inducing chondrogenesis of mesenchymal stem cells

Acta Biomaterialia ◽

10.1016/j.actbio.2014.10.013 ◽

2015 ◽

Vol 12 ◽

pp. 30-41 ◽

Cited By ~ 31

Author(s):

Bogyu Choi ◽

Soyon Kim ◽

Brian Lin ◽

Kevin Li ◽

Olga Bezouglaia ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Visible Light ◽

Extracellular Signaling

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Synthesis and glycosylation of CD52, the major 'maturation-associated' antigen of rat spermatozoa, in the cauda epididymidis

Reproduction ◽

10.1530/rep.0.1210435 ◽

2001 ◽

pp. 435-446 ◽

Cited By ~ 10

Author(s):

P Derr ◽

CH Yeung ◽

TG Cooper ◽

C Kirchhoff

Keyword(s):

Blot Analysis ◽

Cdna Sequence ◽

Northern Blot Analysis ◽

Lectin Binding ◽

Transcriptional Level ◽

Lectin Blot ◽

Molecular Masses ◽

Rat Epididymis ◽

Sperm Membrane ◽

Cauda Epididymidis

A western and lectin blot analysis was performed of the major 'maturation-associated' antigen of rat spermatozoa, which is the rat counterpart of human CD52. In the absence of a suitable antibody, direct study of this approximately 26 kDa antigen, named previously SMemG, had been difficult. In the present study, these problems were overcome by raising a polyclonal antibody against a chemosynthetic peptide predicted from the cDNA sequence of the antigen. The antibody bound to a glycoprotein of rat cauda epididymidal tissue and spermatozoa, this glycoprotein was cleaved by phosphatidylinositol-specific phospholipase C and, after deglycosylation, was reduced to approximately 6 kDa. Northern blot analysis confirmed that the CD52 mRNA was transcribed only post-testicularly, and antibody binding to testicular and sperm proteins of different molecular masses was shown to be nonspecific. Flow cytometry also indicated that the antigen was inserted into the sperm membrane during epididymal transit. Moreover, despite the presence of CD52 mRNA in all parts of the rat epididymis, only the 'long' mRNA molecules of the cauda region were efficiently translated and the antigen glycosylated, indicating that expression of rat CD52 is regulated on a post-transcriptional level. Lectin binding and deglycosylation studies supported the contention that there is extensive mucin-type O-glycosylation of rat CD52. In rats, there was no indication of complex N-linked carbohydrates similar to those described for human CD52.

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Dynamic Interactions and the Evolutionary Genetics of Dental Patterning

Critical Reviews in Oral Biology & Medicine ◽

10.1177/10454411980090040101 ◽

1998 ◽

Vol 9 (4) ◽

pp. 369-398 ◽

Cited By ~ 64

Author(s):

K.M. Weiss ◽

D.W. Stock ◽

Z. Zhao

Keyword(s):

Morphological Structure ◽

Homeobox Gene ◽

Evolutionary Genetics ◽

Gene Families ◽

Developmental Genetics ◽

Dynamic Interactions ◽

Extracellular Signaling ◽

Combinatorial Codes ◽

Tooth Type ◽

Mammalian Teeth

The mammalian dentition is a segmental, or periodically arranged, organ system whose components are arrayed in specific number and in regionally differentiated locations along the linear axes of the jaws. This arrangement evolved from simpler dentitions comprised of many single-cusp teeth of relatively indeterminate number. The different types of mammalian teeth have subsequently evolved as largely independent units. The experimentally documented developmental autonomy of dental primordia shows that the basic dental pattern is established early in embryogenesis. An understanding of how genetic patterning processes may work must be consistent with the different modes of development, and partially independent evolution, of the upper and lower dentition in mammals. The periodic nature of the location, number, and morphological structure of teeth suggests that processes involving the quantitative interaction of diffusible signaling factors may be involved. Several extracellular signaling molecules and their interactions have been identified that may be responsible for locating teeth along the jaws and for the formation of the incisor field. Similarly, the wavelike expression of signaling factors within developing teeth suggests that dynamic interactions among those factors may be responsible for crown patterns. These factors seem to be similar among different tooth types, but the extent to which crown differences can be explained strictly in terms of variation in the parameters of interactions among the same genes, as opposed to tooth-type-specific combinatorial codes of gene expression, is not yet known. There is evidence that combinatorial expression of intracellular transcription factors, including homeobox gene families, may establish domains within the jaws in which different tooth types are able to develop. An evolutionary perspective can be important for our understanding of dental patterning and the designing of appropriate experimental approaches, but dental patterns also raise basic unresolved questions about the nature of the evolutionary assumptions made in developmental genetics.

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Lectin-blot analyses of Trichinella spiralis muscle larvae excretory-secretory components

Parasitology Research ◽

10.1007/s00436-002-0606-7 ◽

2002 ◽

Vol 88 (11) ◽

pp. 1004-1007 ◽

Cited By ~ 22

Author(s):

Alisa Gruden-Movsesijan ◽

Natasa Ilic ◽

Ljiljana Sofronic-Milosavljevic

Keyword(s):

Trichinella Spiralis ◽

Muscle Larvae ◽

Lectin Blot

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Nuclear fibroblast growth factor-2 interacts specifically with splicing factor SF3a66

Biological Chemistry ◽

10.1515/bc.2004.156 ◽

2004 ◽

Vol 385 (12) ◽

pp. 1203-1208 ◽

Cited By ~ 13

Author(s):

Susanne Gringel ◽

Jeroen van Bergeijk ◽

Kirsten Haastert ◽

Claudia Grothe ◽

Peter Claus

Keyword(s):

Growth Factor ◽

Fibroblast Growth Factor ◽

Splicing Factor ◽

Fibroblast Growth Factor 2 ◽

Interacting Proteins ◽

Fibroblast Growth ◽

Extracellular Signaling ◽

C Terminus ◽

Assembly Factor ◽

Smn Protein

Abstract Fibroblast growth factor 2 (FGF-2) has a dual role as a classical extracellular signaling protein and as an intracellular factor. Isoforms of FGF-2, resulting from alternatively used start codons on one mRNA species, locate differentially to nuclear compartments. In this study we aimed to analyze functions of intracellular FGF-2 by identification of interacting proteins. We identified the 66-kDa subunit of splicing factor 3a (SF3a66) as a binding partner in a yeast two-hybrid screen and confirmed this interaction by pull-down assays. The splicing factor interacted with the 18-kDa (FGF-218) and with the 23-kDa (FGF-223) isoforms, indicating an interaction with a domain common to both isoforms. Moreover, FGF-2 interacted with the C-terminus of SF3a66, a sequence that has not previously been assigned a functional role. In a functional neurite outgrowth assay, SF3a66 enhanced neurite lengths similar to FGF-218. We have previously identified the spliceosomal assembly factor survival of motoneuron (SMN) protein as a protein interacting specifically with the FGF-223 isoform [Claus et al., J. Biol. Chem. 278 (2003), 479–485]. The identification of two FGF-2 interacting proteins from the same biochemical pathway suggests a novel intranuclear role of FGF-2.

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