scholarly journals Species-specific pharmacology of maximakinin, an amphibian homologue of bradykinin: putative prodrug activity at the human B2 receptor and peptidase resistance in rat

2016 ◽  
Author(s):  
Xavier Charest-Morin ◽  
Hélène Bachelard ◽  
Melissa Jean ◽  
Francois Marceau
Keyword(s):  
Mast Cell ◽  
Fluorescent Protein ◽  
Radioligand Binding ◽  
Umbilical Vein ◽  
Mammalian Species ◽  
Cell Secretion ◽  
Human Form ◽  
Species Specific ◽  
In Cells

Maximakinin (MK), an amphibian peptide possessing the C-terminal sequence of bradykinin (BK), is a BK B2 receptor (B2R) agonist eliciting prolonged signaling. We reinvestigated this 19-mer for species-specific pharmacologic profile, in vivo confirmation of resistance to inactivation by angiotensin converting enzyme (ACE), value as a module for the design of fusion proteins that bind to the B2R in mammalian species and potential activity as a histamine releaser. Competition of the binding of [3H]BK to recombinant human myc-B2Rs in cells that express these receptors revealed that MK possessed a tenuous fraction (<0.1%) of the affinity of BK, despite being only ~10-fold less potent than BK in a contractility assay based on the human isolated umbilical vein. These findings are reconciled by the generation of C-terminal fragments, like Lys-Gly-Pro-BK and Gly-Pro-BK, when the latent MK is incubated with human venous tissue (LC-MS), supporting activation via hydrolysis upstream of the BK sequence. At the rat recombinant myc-B2R, MK had a lesser affinity than that of BK, but with a narrower margin (6.2-fold, radioligand binding competition). Accordingly, MK (10 nM) stimulated calcium transients in cells that expressed the rat receptors, but not the human B2R. Recombinant MRGPRX2, a receptor that mediates cationic peptide-induced mast cell secretion, minimally responded by increased [Ca+2]i to MK at 10 μM. Enhanced green fluorescent protein fused to MK (EGFP-MK) labeled cells that expressed rat, but not human B2Rs. Intravenous MK induced dose-dependent hypotensive, vasodilator and tachycardic responses in anesthetized rats and the effects were antagonized by pretreatment with icatibant but not modified by pyrilamine or enalaprilat. Strong species-specific responses to the toxin-derived peptide MK and its prodrug status in the isolated human vein were evidenced. Accordingly, MK in the EGFP-MK fusion protein is a pharmacophore module that confers affinity for the rat B2R, but not for the human form of the B2R. MK is unlikely to be an efficient mast cell activator, but its resistance to inactivation by ACE was confirmed in vivo.

scholarly journals Species-specific pharmacology of maximakinin, an amphibian homologue of bradykinin: putative prodrug activity at the human B2 receptor and peptidase resistance in rat

2016 ◽  
Author(s):  
Xavier Charest-Morin ◽  
Hélène Bachelard ◽  
Melissa Jean ◽  
Francois Marceau
Keyword(s):  
Mast Cell ◽  
Fluorescent Protein ◽  
Radioligand Binding ◽  
Umbilical Vein ◽  
Mammalian Species ◽  
Cell Secretion ◽  
Human Form ◽  
Species Specific ◽  
In Cells

Maximakinin (MK), an amphibian peptide possessing the C-terminal sequence of bradykinin (BK), is a BK B2 receptor (B2R) agonist eliciting prolonged signaling. We reinvestigated this 19-mer for species-specific pharmacologic profile, in vivo confirmation of resistance to inactivation by angiotensin converting enzyme (ACE), value as a module for the design of fusion proteins that bind to the B2R in mammalian species and potential activity as a histamine releaser. Competition of the binding of [3H]BK to recombinant human myc-B2Rs in cells that express these receptors revealed that MK possessed a tenuous fraction (<0.1%) of the affinity of BK, despite being only ~10-fold less potent than BK in a contractility assay based on the human isolated umbilical vein. These findings are reconciled by the generation of C-terminal fragments, like Lys-Gly-Pro-BK and Gly-Pro-BK, when the latent MK is incubated with human venous tissue (LC-MS), supporting activation via hydrolysis upstream of the BK sequence. At the rat recombinant myc-B2R, MK had a lesser affinity than that of BK, but with a narrower margin (6.2-fold, radioligand binding competition). Accordingly, MK (10 nM) stimulated calcium transients in cells that expressed the rat receptors, but not the human B2R. Recombinant MRGPRX2, a receptor that mediates cationic peptide-induced mast cell secretion, minimally responded by increased [Ca+2]i to MK at 10 μM. Enhanced green fluorescent protein fused to MK (EGFP-MK) labeled cells that expressed rat, but not human B2Rs. Intravenous MK induced dose-dependent hypotensive, vasodilator and tachycardic responses in anesthetized rats and the effects were antagonized by pretreatment with icatibant but not modified by pyrilamine or enalaprilat. Strong species-specific responses to the toxin-derived peptide MK and its prodrug status in the isolated human vein were evidenced. Accordingly, MK in the EGFP-MK fusion protein is a pharmacophore module that confers affinity for the rat B2R, but not for the human form of the B2R. MK is unlikely to be an efficient mast cell activator, but its resistance to inactivation by ACE was confirmed in vivo.

scholarly journals Species-specific pharmacology of maximakinin, an amphibian homologue of bradykinin: putative prodrug activity at the human B2receptor and peptidase resistance in rats

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e2911 ◽  
Author(s):  
Xavier Charest-Morin ◽  
Hélène Bachelard ◽  
Melissa Jean ◽  
Francois Marceau
Keyword(s):  
Mast Cell ◽  
Fluorescent Protein ◽  
Radioligand Binding ◽  
Umbilical Vein ◽  
Mammalian Species ◽  
Cell Secretion ◽  
Human Form ◽  
Species Specific ◽  
In Cells

Maximakinin (MK), an amphibian peptide possessing the C-terminal sequence of bradykinin (BK), is a BK B2receptor (B2R) agonist eliciting prolonged signaling. We reinvestigated this 19-mer for species-specific pharmacologic profile,in vivoconfirmation of resistance to inactivation by angiotensin converting enzyme (ACE), value as a module for the design of fusion proteins that bind to the B2R in mammalian species and potential activity as a histamine releaser. Competition of the binding of [3H]BK to recombinant human myc-B2Rs in cells that express these receptors revealed that MK possessed a tenuous fraction (<0.1%) of the affinity of BK, despite being only ∼20-fold less potent than BK in a contractility assay based on the human isolated umbilical vein. These findings are reconciled by the generation of C-terminal fragments, like Lys-Gly-Pro-BK and Gly-Pro-BK, when the latent MK is incubated with human venous tissue (LC-MS), supporting activationviahydrolysis upstream of the BK sequence. At the rat recombinant myc-B2R, MK had a lesser affinity than that of BK, but with a narrower margin (6.2-fold, radioligand binding competition). Accordingly, MK (10 nM) stimulated calcium transients in cells that expressed the rat receptors, but not the human B2R. Recombinant MRGPRX2, a receptor that mediates cationic peptide-induced mast cell secretion, minimally responded by increased [Ca+2]ito MK at 10 µM. Enhanced green fluorescent protein fused to MK (EGFP-MK) labeled cells that expressed rat, but not human B2Rs. Intravenous MK induced dose-dependent hypotensive, vasodilator and tachycardic responses in anesthetized rats and the effects were antagonized by pretreatment with icatibant but not modified by pyrilamine or enalaprilat. Strong species-specific responses to the toxin-derived peptide MK and its prodrug status in the isolated human vein were evidenced. Accordingly, MK in the EGFP-MK fusion protein is a pharmacophore module that confers affinity for the rat B2R, but not for the human form of the B2R. MK is unlikely to be an efficient mast cell activator, but its resistance to inactivation by ACE was confirmedin vivo.

Nerve-mast cell (RBL) interaction: RBL membrane ruffling occurs at the contact site with an activated neurite

2002 ◽  
Vol 283 (6) ◽  
pp. C1738-C1744 ◽  
Author(s):  
N. Mori ◽  
R. Suzuki ◽  
T. Furuno ◽  
D. M. McKay ◽  
M. Wada ◽  
...  
Keyword(s):  
Mast Cell ◽  
Cell Activation ◽  
Fluorescent Protein ◽  
Cell Communication ◽  
Scorpion Venom ◽  
Confocal Imaging ◽  
Mucosal Mast Cell ◽  
Contact Site ◽  
Membrane Ruffling

Mast cell-neurite interaction serves as a model for neuroimmune interaction. We have shown that neurite-mast cell communication can occur via substance P interacting with neurokinin (NK)-1 receptors on the mucosal mast cell-like cell, the rat basophilic leukemia (RBL) cell. Neurite (murine superior cervical ganglia) and RBL cell [expressing the granule-associated antigen CD63-green fluorescent protein (GFP) conjugate] cocultures were established and stimulated with bradykinin (BK; 10 nM) or scorpion venom (SV; 10 pg/ml), both of which activate only neurites. Cell activation was assessed by confocal imaging of Ca2+ (cells preloaded with fluo 3), and analyses of RBL CD63-GFP+ granule movement were conducted. Neurite activation by BK or SV was followed by RBL Ca2+mobilization, which was inhibited by an NK-1 receptor antagonist (NK-1 RA). Moreover, membrane ruffling was observed on RBL pseudopodial extensions in contact with the activated neurite, but not on noncontacting pseudopodia. RBL membrane ruffling was inhibited by NK-1 RA, but not NK-2 RA, and was accompanied by a significant increase in granule movement (0.13 ± 0.04 vs. 0.05 ± 0.01 μm/s) that was most evident at the point of neurite contact: many of the granules moved toward the plasmalemma. This is the first documentation of such precise (restricted to the membrane's contact site) transfer of information between nerves and mast cells that could allow for very subtle in vivo communication between these two cell types.

scholarly journals Pleiotropic nuclear defects associated with a conditional allele of the novel nucleoporin Rat9p/Nup85p.

1996 ◽  
Vol 7 (6) ◽  
pp. 917-934 ◽  
Author(s):  
A L Goldstein ◽  
C A Snay ◽  
C V Heath ◽  
C N Cole
Keyword(s):  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Main Body ◽  
The Novel ◽  
Conditional Allele ◽  
Green Fluorescent ◽  
Nucleocytoplasmic Export ◽  
In Cells

In a screen for mutants defective in nucleocytoplasmic export of mRNA, we have identified a new component of the Saccharomyces cerevisiae nuclear pore complex (NPC). The RAT9/NUP85 (ribonucleic acid trafficking) gene encodes an 84.9-kDa protein that we have localized to NPCs by tagging the RAT9/NUP85 gene with the in vivo molecular marker Green Fluorescent Protein. In cells containing either the rat9-1 allele or a complete deletion of the RAT9/NUP85 gene, poly(A)+ RNA accumulates rapidly in nuclei after a shift from 23 degrees C to 37 degrees C. Under these same conditions, rapid fragmentation of the nucleolus was also observed. At the permissive growth temperature in rat9-1 or RAT9 deletion strains, the nuclear envelope (NE) becomes detached from the main body of the nucleus, forming long thin double sheets of NE. NPCs within these sheets are clustered and some appear to be locked together between opposing sheets of NE such that their nucleoplasmic faces are in contact. The Rat9/Nup85 protein could not be detected in cells carrying a mutation of RAT2/NUP120, suggesting that Rat9p/Nup85p cannot be assembled into NPCs in the absence of Rat2p/Nup120p. In contrast,Rat9/ Nup85 protein was readily incorporated into NPCs in strains carrying mutant alleles of other nucleoporin genes. The possible role of Rat9p/Nup85p in NE integrity and the loss of nucleoporins when another nucleoporin is mutant or absent are discussed.

scholarly journals Circulating Skeletal Stem Cells

2001 ◽  
Vol 153 (5) ◽  
pp. 1133-1140 ◽  
Author(s):  
Sergei A. Kuznetsov ◽  
Mahesh H. Mankani ◽  
Stan Gronthos ◽  
Kazuhito Satomura ◽  
Paolo Bianco ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Cells ◽  
Mammalian Species ◽  
Osteogenic Potential ◽  
Single Colony ◽  
Transplantation Assay ◽  
Immunocompromised Mice ◽  
Species Specific ◽  
Stromal Stem Cells

We report the isolation of adherent, clonogenic, fibroblast-like cells with osteogenic and adipogenic potential from the blood of four mammalian species. These cells phenotypically resemble but are distinguishable from skeletal stem cells found in bone marrow (stromal stem cells, “mesenchymal stem cells”). The osteogenic potential of the blood-borne cells was proven by an in vivo transplantation assay in which either polyclonal or single colony–derived strains were transplanted into the subcutis of immunocompromised mice, and the donor origin of the fully differentiated bone cells was proven using species-specific probes. This is the first definitive proof of the existence of circulating skeletal stem cells in mammals.

scholarly journals A Robust Bioassay of the Human Bradykinin B2 Receptor that Extends Molecular and Cellular Studies: The Isolated Umbilical Vein

2021 ◽  
Vol 14 (3) ◽  
pp. 177
Author(s):  
François Marceau ◽  
Hélène Bachelard
Keyword(s):  
Partial Agonist ◽  
Umbilical Vein ◽  
In Vivo Studies ◽  
Molecular Pharmacology ◽  
Smooth Muscle Contractility ◽  
Competitive Antagonist ◽  
Pharmacological Agents ◽  
Species Specific ◽  
G Protein Coupled

Bradykinin (BK) has various physiological and pathological roles. Medicinal chemistry efforts targeted toward the widely expressed BK B2 receptor (B2R), a G-protein-coupled receptor, were primarily aimed at developing antagonists. The only B2R antagonist in clinical use is the peptide icatibant, approved to abort attacks of hereditary angioedema. However, the anti-inflammatory applications of B2R antagonists are potentially wider. Furthermore, the B2R antagonists notoriously exhibit species-specific pharmacological profiles. Classical smooth muscle contractility assays are exploited over a time scale of several hours and support determining potency, competitiveness, residual agonist activity, specificity, and reversibility of pharmacological agents. The contractility assay based on the isolated human umbilical vein, expressing B2R at physiological density, was introduced when investigating the first non-peptide B2R antagonist (WIN 64338). Small ligand molecules characterized using the assay include the exquisitely potent competitive antagonist, Pharvaris Compound 3 or the partial agonist Fujisawa Compound 47a. The umbilical vein assay is also useful to verify pharmacologic properties of special peptide B2R ligands, such as the carboxypeptidase-activated latent agonists and fluorescent probes. Furthermore, the proposed agonist effect of tissue kallikrein on the B2R has been disproved using the vein. This assay stands in between cellular and molecular pharmacology and in vivo studies.

scholarly journals Gene Therapy: A Lipofection Approach for Gene Transfer into Primary Endothelial Cells

2002 ◽  
Vol 11 (6) ◽  
pp. 573-582 ◽  
Author(s):  
A. T. L. Young ◽  
J. R. T. Lakey ◽  
A. G. Murray ◽  
R. B. Moore
Keyword(s):  
Gene Therapy ◽  
Endothelial Cells ◽  
Gene Delivery ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Umbilical Vein ◽  
Vector System ◽  
Microscopic Evaluation

Despite the great potential of gene therapy to become a new treatment modality in future medicine, there are still many limitations to overcome before this gene approach can pass to the stage of human trial. The foremost obstacle is the development of a safe, efficient, and efficacious vector system for in vivo gene application. This study evaluated the efficacy of lipofection as a gene delivery vehicle into primary endothelial cells. Transfection efficiency of several lipid-based reagents (Effectene, Fugene 6, DOTAP) was examined at experimental temperatures of 37°C, 24°C, and 6°C. Human umbilical vein endothelial cells (HUVECs) were transfected with the enhanced green fluorescent protein (EGFP) using precise amounts of DNA (Effectene, 0.2 μg; Fugene 6, 0.5 μg; DOTAP, 2.5 μg) and lipids (Effectene, 10 μl; Fugene 6, 6 μl; DOTAP, 15 μl) optimized in our laboratory. Duration of incubation in the DNA/lipid transfection mixture varied for each lipid transfectant as follows: 5 h for both Fugene 6 and DOTAP and 3 h for Effectene. Efficiency of transfection was quantified by microscopic evaluation of EFGP expression in a minimum of 100 cells per group. Transfection efficiencies achieved with these lipofection agents were 34 ± 1.3% (mean ± SEM), 33 ± 1.4%, and 18 ± 1.5% for Effectene, Fugene 6, and DOTAP, respectively, at 37°C. Transfection results were lower at 24°C with mean efficiencies of 26 ± 2.4% for Effectene, 14 ± 2.9% for Fugene 6, and 15 ± 3.2% for DOTAP. Furthermore, mean efficiencies at 6°C were 6 ± 0.5%, 8 ± 1.5%, and 6 ± 0.0% for Effectene, Fugene 6, and DOTAP, respectively. Efficiency of transfection appeared to be temperature dependent (ANOVA; p < 0.0001). In spite of a significant decrease (37°C vs. 24°C: p < 0.0001; 37°C vs. 6°C: p < 0.0001; 24°C vs. 6°C: p < 0.0115) in transfection efficiency at low temperatures, the successful in vitro gene manipulation renders lipofection a potential gene delivery strategy for in vivo gene therapy.

scholarly journals Coaxially Bioprinted Cell-Laden Tubular-Like Structure for Studying Glioma Angiogenesis

2021 ◽  
Vol 9 ◽  
Author(s):  
Xuanzhi Wang ◽  
Xinda Li ◽  
Yi Zhang ◽  
Xiaoyan Long ◽  
Haitao Zhang ◽  
...  
Keyword(s):  
Growth Factor ◽  
Blood Vessels ◽  
Tumor Angiogenesis ◽  
Fluorescent Protein ◽  
Umbilical Vein ◽  
Three Dimensional ◽  
Tumor Model ◽  
3D Models ◽  
3D Microenvironment

Glioblastomas are the most frequently diagnosed and one of the most lethal primary brain tumors, and one of their key features is a dysplastic vascular network. However, because the origin of the tumor blood vessels remains controversial, an optimal preclinical tumor model must be established to elucidate the tumor angiogenesis mechanism, especially the role of tumor cells themselves in angiogenesis. Therefore, shell-glioma cell (U118)-red fluorescent protein (RFP)/core-human umbilical vein endothelial cell (HUVEC)-green fluorescent protein (GFP) hydrogel microfibers were coaxially bioprinted. U118–RFP and HUVEC–GFP cells both exhibited good proliferation in a three-dimensional (3D) microenvironment. The secretability of both vascular endothelial growth factor A and basic fibroblast growth factor was remarkably enhanced when both types of cells were cocultured in 3D models. Moreover, U118 cells promoted the vascularization of the surrounding HUVECs by secreting vascular growth factors. More importantly, U118–HUVEC-fused cells were found in U118–RFP/HUVEC–GFP hydrogel microfibers. Most importantly, our results indicated that U118 cells can not only recruit the blood vessels of the surrounding host but also directly transdifferentiate into or fuse with endothelial cells to participate in tumor angiogenesis in vivo. The coaxially bioprinted U118–RFP/HUVEC–GFP hydrogel microfiber is a model suitable for mimicking the glioma microenvironment and for investigating tumor angiogenesis.

scholarly journals On and Off Membrane Dynamics of the Endoplasmic Reticulum–Golgi Tethering Factor p115 In Vivo

2006 ◽  
Vol 17 (7) ◽  
pp. 2996-3008 ◽  
Author(s):  
Elizabeth Brandon ◽  
Tomasz Szul ◽  
Cecilia Alvarez ◽  
Robert Grabski ◽  
Ronald Benjamin ◽  
...  
Keyword(s):  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Membrane Dynamics ◽  
Snare Complex ◽  
Fluorescence Recovery ◽  
Cell Periphery ◽  
Vesicular Traffic ◽  
Kinetics Of ◽  
In Cells

The mechanisms regulating membrane recruitment of the p115 tethering factor in vivo are unknown. Here, we describe cycling of p115 between membranes and cytosol and document the effects of Golgi matrix proteins, Rab1, and soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) on this process. Rapid membrane/cytosol exchange is shown by swift (t1/2 ∼20 s) loss of Golgi-localized p115-green fluorescent protein (GFP) after repeated photobleaching of cell periphery and rapid (t1/2 ∼13 s) fluorescence recovery after photobleaching Golgi-localized p115-GFP. p115 mutant missing the GM130/giantin binding site exhibits analogous fluorescence recovery after photobleaching (FRAP) (t1/2 ∼13 s), suggesting that GM130 and giantin are not major determinants of p115 membrane dynamics. In contrast, p115-GFP exchanges more rapidly (t1/2 ∼8 s) in cells expressing the inactive Rab1/N121I mutant, indicating that p115 cycling is influenced by Rab1. p115-GFP dynamics is also influenced by the assembly status of SNAREs. In cells expressing an ATPase-deficient NSF/E329Q mutant that inhibits SNARE complex disassembly, the cycling kinetics of p115-GFP are significantly slower (t1/2 ∼21 s). In contrast, in cells incubated at reduced temperature (10°C) that inhibits vesicular traffic, the cycling kinetics of p115-GFP are faster (t1/2 ∼7 s). These data suggest that p115-binding sites on the membrane are provided by unassembled SNAREs. In agreement, biochemical studies show increased p115 recruitment to membranes in the presence of NSF and α-SNAP. Our data support a model in which recruitment of tethers is directly regulated by the assembly status of SNAREs.

Effect of Some Metabolic Inhibitors on Vinblastine-Induced Ribosomal-Granular Material Complexes

1971 ◽  
Vol 29 ◽  
pp. 548-549
Author(s):  
Awtar Krishan ◽  
Dora Hsu
Keyword(s):  
Granular Material ◽  
Rna Synthesis ◽  
Culture Medium ◽  
Actinomycin D ◽  
Metabolic Inhibitors ◽  
Protein And Rna Synthesis ◽  
Apparent Reduction ◽  
Plant Alkaloids ◽  
In Cells

Cells exposed to antitumor plant alkaloids, vinblastine and vincristine sulfate have large proteinacious crystals and complexes of ribosomes, helical polyribosomes and electron-dense granular material (ribosomal complexes) in their cytoplasm, Binding of H3-colchicine by the in vivo crystals shows that they contain microtubular proteins. Association of ribosomal complexes with the crystals suggests that these structures may be interrelated.In the present study cultured human leukemic lymphoblasts (CCRF-CEM), were incubated with protein and RNA-synthesis inhibitors, p. fluorophenylalanine, puromycin, cycloheximide or actinomycin-D before the addition of crystal-inducing doses of vinblastine to the culture medium. None of these compounds could completely prevent the formation of the ribosomal complexes or the crystals. However, in cells pre-incubated with puromycin, cycloheximide, or actinomycin-D, a reduction in the number and size of the ribosomal complexes was seen. Large helical polyribosomes were absent in the ribosomal complexes of cells treated with puromycin, while in cells exposed to cycloheximide, there was an apparent reduction in the number of ribosomes associated with the ribosomal complexes (Fig. 2).

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