scholarly journals Role of SP65 in Assembly of the Dictyostelium discoideum Spore Coat

2007 ◽  
Vol 6 (7) ◽  
pp. 1137-1149 ◽  
Author(s):  
Talibah Metcalf ◽  
Hanke van der Wel ◽  
Ricardo Escalante ◽  
Leandro Sastre ◽  
Christopher M. West
Keyword(s):  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Outer Layer ◽  
Fluorescent Protein ◽  
De Novo ◽  
Middle Layer ◽  
In Vivo Studies ◽  
Permeability Barrier ◽  
Spore Coat

ABSTRACT Like the cyst walls of other protists, the spore coat of Dictyostelium discoideum is formed de novo to protect the enclosed dormant cell from stress. Spore coat assembly is initiated by exocytosis of protein and polysaccharide precursors at the cell surface, followed by the infusion of nascent cellulose fibrils, resulting in an asymmetrical trilaminar sandwich with cellulose filling the middle layer. A molecular complex consisting of cellulose and two proteins, SP85 and SP65, is associated with the inner and middle layers and is required for proper organization of distinct proteins in the outer layer. Here we show that, unlike SP85 and other protein precursors, which are stored in prespore vesicles, SP65 is, like cellulose, synthesized just in time. By tagging the SP65 locus with green fluorescent protein, we find that SP65 is delivered to the cell surface via largely distinct vesicles, suggesting that separate delivery of components of the cellulose-SP85-SP65 complex regulates its formation at the cell surface. In support of previous in vivo studies, recombinant SP65 and SP85 are shown to interact directly. In addition, truncation of SP65 causes a defect of the outer layer permeability barrier as seen previously for SP85 mutants. These observations suggest that assembly of the cellulose-SP85-SP65 triad at the cell surface is biosynthetically regulated both temporally and spatially and that the complex contributes an essential function to outer layer architecture and function.

scholarly journals Helminth secretions induce de novo T cell Foxp3 expression and regulatory function through the TGF-β pathway

2010 ◽  
Vol 207 (11) ◽  
pp. 2331-2341 ◽  
Author(s):  
John R. Grainger ◽  
Katie A. Smith ◽  
James P. Hewitson ◽  
Henry J. McSorley ◽  
Yvonne Harcus ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Fluorescent Protein ◽  
De Novo ◽  
Foxp3 Expression ◽  
Dominant Negative ◽  
Regulatory Function ◽  
Intestinal Helminth ◽  
Helminth Parasites

Foxp3-expressing regulatory T (T reg) cells have been implicated in parasite-driven inhibition of host immunity during chronic infection. We addressed whether parasites can directly induce T reg cells. Foxp3 expression was stimulated in naive Foxp3− T cells in mice infected with the intestinal helminth Heligmosomoides polygyrus. In vitro, parasite-secreted proteins (termed H. polygyrus excretory-secretory antigen [HES]) induced de novo Foxp3 expression in fluorescence-sorted Foxp3− splenocytes from Foxp3–green fluorescent protein reporter mice. HES-induced T reg cells suppressed both in vitro effector cell proliferation and in vivo allergic airway inflammation. HES ligated the transforming growth factor (TGF) β receptor and promoted Smad2/3 phosphorylation. Foxp3 induction by HES was lost in dominant-negative TGF-βRII cells and was abolished by the TGF-β signaling inhibitor SB431542. This inhibitor also reduced worm burdens in H. polygyrus–infected mice. HES induced IL-17 in the presence of IL-6 but did not promote Th1 or Th2 development under any conditions. Importantly, antibody to mammalian TGF-β did not recognize HES, whereas antisera that inhibited HES did not affect TGF-β. Foxp3 was also induced by secreted products of Teladorsagia circumcincta, a related nematode which is widespread in ruminant animals. We have therefore identified a novel pathway through which helminth parasites may stimulate T reg cells, which is likely to be a key part of the parasite’s immunological relationship with the host.

scholarly journals Targeting Mucosal Sites by Polymeric Immunoglobulin Receptor-directed Peptides

2002 ◽  
Vol 196 (4) ◽  
pp. 551-555 ◽  
Author(s):  
Kendra D. White ◽  
J. Donald Capra
Keyword(s):  
Epithelial Cells ◽  
Fluorescent Protein ◽  
Secretory Component ◽  
In Vivo Studies ◽  
Polymeric Immunoglobulin Receptor ◽  
Immunoglobulin Receptor ◽  
Potential Binding Site ◽  
Green Fluorescent

Polymeric immunoglobulins provide first line humoral defense at mucosal surfaces to which they are specifically transported by the polymeric immunoglobulin receptor (pIgR) on mucosal and glandular epithelial cells. Previous studies from our laboratory suggested that amino acids 402–410 of the Cα3 domain of dimeric IgA (dIgA) represented a potential binding site for the pIgR. Here by binding human secretory component to overlapping decapeptides of Cα3, we confirm these residues and also uncover an additional site. Furthermore, we show that the Cα3 motif appears to be sufficient to direct transport of green fluorescent protein through the pIgR-specific cellular transcytosis system. An alternative approach identified phage peptides, selected from a library by the in vitro Madin Darby Canine Kidney transcytosis assay, for pIgR-mediated transport through epithelial cells. Some transcytosis-selected peptides map to the same 402–410 pIgR-binding Cα3 site. Further in vivo studies document that at least one of these peptides is transported in a rat model measuring hepatic bile transport. In addition to identifying small peptides that are both bound and transported by the pIgR, this study provides evidence that the pIgR-mediated mucosal secretion system may represent a means of targeting small molecule therapeutics and genes to mucosal epithelial cells.

scholarly journals Normal mouse peritoneum contains a large population of Ly-1+ (CD5) B cells that recognize phosphatidyl choline. Relationship to cells that secrete hemolytic antibody specific for autologous erythrocytes.

1988 ◽  
Vol 168 (2) ◽  
pp. 687-698 ◽  
Author(s):  
T J Mercolino ◽  
L W Arnold ◽  
L A Hawkins ◽  
G Haughton
Keyword(s):  
B Cells ◽  
Cell Surface ◽  
Phosphatidyl Choline ◽  
Large Population ◽  
In Vivo Studies ◽  
Antigen Specificity ◽  
Adult Mice ◽  
Large Numbers

We have found that, in the peritoneums of normal adult mice, 5-15% of lymphocytes bind a fluorescent liposome probe. In ontogeny, cells with this specificity were shown to appear by 8 d after birth, and increase to the adult frequency by 2-3 wk. Some older mice contain an expanded population of these cells. We have shown that liposome binding occurs by cell surface IgM recognizing the common membrane phospholipid, phosphatidyl choline (PtC). Virtually all of these PtC-specific cells bear the cell surface marker Ly-1. Our results indicate that roughly 1 in 10 peritoneal Ly-1+ B cells has this single specificity. We have found that the precursors to all the cells that form plaques on protease-treated autologous erythrocytes (BrMRBC) are included in the PtC-specific population and can be isolated by FACS. We believe this is the first report of sorting large numbers of B cells with a single antigen specificity from normal, unimmunized animals. This method will allow for in vitro and in vivo studies of differentiative and proliferative properties of Ly-1+ B cells, which may help define their role in development and disease.

scholarly journals Direct Interactions between Calcitonin-Like Receptor (CLR) and CGRP-Receptor Component Protein (RCP) Regulate CGRP Receptor Signaling

Endocrinology ◽  
2012 ◽  
Vol 153 (4) ◽  
pp. 1850-1860 ◽  
Author(s):  
Sophie C. Egea ◽  
Ian M. Dickerson
Keyword(s):  
Cell Surface ◽  
Transmembrane Protein ◽  
Direct Interaction ◽  
Dominant Negative ◽  
In Vivo Studies ◽  
Cgrp Receptor ◽  
Receptor Component ◽  
Soluble Fusion Protein ◽  
Component Protein

Calcitonin gene-related peptide (CGRP) is a neuropeptide with multiple neuroendocrine roles, including vasodilation, migraine, and pain. The receptor for CGRP is a G protein-coupled receptor (GPCR) that requires three proteins for function. CGRP binds to a heterodimer composed of the GPCR calcitonin-like receptor (CLR) and receptor activity-modifying protein (RAMP1), a single transmembrane protein required for pharmacological specificity and trafficking of the CLR/RAMP1 complex to the cell surface. In addition, the CLR/RAMP1 complex requires a third protein named CGRP-receptor component protein (RCP) for signaling. Previous studies have demonstrated that depletion of RCP from cells inhibits CLR signaling, and in vivo studies have demonstrated that expression of RCP correlates with CLR signaling and CGRP efficacy. It is not known whether RCP interacts directly with CLR to exert its effect. The current studies identified a direct interaction between RCP and an intracellular domain of CLR using yeast two-hybrid analysis and coimmunoprecipitation. When this interacting domain of CLR was expressed as a soluble fusion protein, it coimmunoprecipitated with RCP and inhibited signaling from endogenous CLR. Expression of this dominant-negative domain of CLR did not significantly inhibit trafficking of CLR to the cell surface, and thus RCP may not have a chaperone function for CLR. Instead, RCP may regulate CLR signaling in the cell membrane, and direct interaction between RCP and CLR is required for CLR activation. To date, RCP has been found to interact only with CLR and represents a novel neuroendocrine regulatory step in GPCR signaling.

scholarly journals In vivo analysis of human nucleoporin repeat domain interactions

2013 ◽  
Vol 24 (8) ◽  
pp. 1222-1231 ◽  
Author(s):  
Songli Xu ◽  
Maureen A. Powers
Keyword(s):  
Fluorescent Protein ◽  
Live Cells ◽  
Nuclear Pore ◽  
Permeability Barrier ◽  
Nucleocytoplasmic Trafficking ◽  
Repeat Domain ◽  
Domain Interactions ◽  
In Vivo Analysis ◽  
Green Fluorescent

The nuclear pore complex (NPC), assembled from ∼30 proteins termed nucleoporins (Nups), mediates selective nucleocytoplasmic trafficking. A subset of nucleoporins bear a domain with multiple phenylalanine–glycine (FG) motifs. As binding sites for transport receptors, FG Nups are critical in translocation through the NPC. Certain FG Nups are believed to associate via low-affinity, cohesive interactions to form the permeability barrier of the pore, although the form and composition of this functional barrier are debated. We used green fluorescent protein–Nup98/HoxA9 constructs with various numbers of repeats and also substituted FG domains from other nucleoporins for the Nup98 domain to directly compare cohesive interactions in live cells by fluorescence recovery after photobleaching (FRAP). We find that cohesion is a function of both number and type of FG repeats. Glycine–leucine–FG (GLFG) repeat domains are the most cohesive. FG domains from several human nucleoporins showed no interactions in this assay; however, Nup214, with numerous VFG motifs, displayed measurable cohesion by FRAP. The cohesive nature of a human nucleoporin did not necessarily correlate with that of its yeast orthologue. The Nup98 GLFG domain also functions in pore targeting through binding to Nup93, positioning the GLFG domain in the center of the NPC and supporting a role for this nucleoporin in the permeability barrier.

scholarly journals GFP transgenic animals in biomedical research: a review of potential disadvantages

2019 ◽  
pp. 525-530
Author(s):  
N. Lipták ◽  
Z. Bősze ◽  
L. Hiripi
Keyword(s):  
Transgenic Mice ◽  
Biomedical Research ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Transgenic Animals ◽  
In Vivo Studies ◽  
Reporter Protein ◽  
Physiological Processes ◽  
Green Fluorescent

Green Fluorescent protein (GFP) transgenic animals are accepted tools for studying various physiological processes, including organ development and cell migration. However, several in vivo studies claimed that GFP may impair transgenic animals’ health. Glomerulosclerosis was observed in transgenic mice and rabbits with ubiquitous reporter protein expression. Heart-specific GFP expression evoked dilated cardiomyopathy and altered cardiac function in transgenic mouse and zebrafish lines, respectively. Moreover, growth retardation and increased axon swelling were observed in GFP and yellow fluorescent protein (YFP) transgenic mice, respectively. This review will focus on the potential drawbacks of the applications of GFP transgenic animals in biomedical research.

scholarly journals Insects as a source of phenolic compounds and potential health benefits

2021 ◽  
pp. 1-12
Author(s):  
M.C. Nino ◽  
L. Reddivari ◽  
C. Osorio ◽  
I. Kaplan ◽  
A.M. Liceaga
Keyword(s):  
Phenolic Compounds ◽  
De Novo ◽  
Health Benefits ◽  
Antimicrobial Activities ◽  
In Vivo Studies ◽  
Plant Phenolics ◽  
Potential Health ◽  
Bioactive Potential

The use of insects in traditional medicine and unveiling the chemical structure of the bright pigments in butterfly wings led to the discovery of bioactive phenolic compounds in the insect bodies. These metabolites have been found not only due to the insect absorption and metabolisation of the plant-derived phenolic present in their diet, but also from the ability of insects to synthesise phenolic compounds de novo through the sclerotisation process. Plant phenolics are secondary metabolites involved in the protection of tissues against UV radiation, herbivores, and pathogens, as well as pigmentation of fruits and flowers. These bioactive compounds exhibit antioxidant, anti-inflammatory, anticancer, and antimicrobial activities, demonstrated through in vitro and in vivo studies. This bioactive potential is thought to occur due to their chemical characteristics that allow them to stabilise reactive oxygen species (ROS), chelate prooxidant metal ions, interact with key enzymes and signal cascades involved in biological pathways. Bioactivity of plant phenolics and both in vitro, in vivo studies, suggest that the dietary compounds absorbed by the insect maintain their chemical and bioactive properties. Further characterisation of the phenolic composition in edible insects and evaluation of their bioactive capacity as well as their bioavailability, could result in discovering additional health benefits of entomophagy apart from macro-nutritional (e.g. protein) content.

scholarly journals A Novel FBXO45-Gef-H1 Axis Controls Oncogenic Signaling in B-Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 711-711
Author(s):  
Anagh Anant Sahasrabuddhe ◽  
Xiaofei Chen ◽  
Kaiyu Ma ◽  
Rui Wu ◽  
Richa Kapoor ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
De Novo ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
In Vivo Studies

Abstract Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common form of malignant lymphoma and may arise de novo, or through transformation from a pre-existing low-grade B cell lymphoma such as follicular lymphoma (FL). However, the post-translational mechanisms and deregulated pathways underlying the pathogenesis of disease evolution are not fully understood. Methods: We employed integrated functional and structural genomics and mass spectrometry (MS)-driven proteomics which implicated a possible novel tumor suppressor role for a conserved E3 ubiquitin ligase FBXO45 in DLBCL pathogenesis. We generated conditional knockout mice targeting loss of Fbxo45 in germinal center (GC) B-cells using the Cg1-Cre-loxP system and an assortment of CRISPR-mediated knockouts of FBXO45 in B cell lymphoma cells (FL518, BJAB, U2932). We engineered B cell lines (BJAB, U2932) to inducibly express FLAG-tagged FBXO45 to identify candidate substrates of FBXO45 using liquid chromatography-tandem MS. In vitro biochemical and in vivo studies using a variety of genetically-modified lines in xenograft studies in immunodeficient mice were performed to validate observations from proteogenomic studies. Whole genome sequencing (WGS) and genomic copy number studies were interrogated to investigate structural alterations targeting FBXO45 in primary human lymphoma samples. Results: Conditional targeting of Fbxo45 in GCB-cells in transgenic mice resulted in abnormal germinal center formation with increased number and size of germinal centers. Strikingly, targeted deletion of Fbxo45 in GCB-cells resulted in spontaneous B cell lymphomas with (22/22);100%) penetrance and none of the wild-type (WT) littermates (0/20; 0%) developed lymphoma at 24 months. Macroscopic examination revealed large tumor masses, splenomegaly, and lymphadenopathy at different anatomic locations including ileocecal junction, mesenteric, retroperitoneal and cervical lymph nodes and thymus. Next generation sequencing of immunoglobulin heavy chain genes revealed monoclonal or oligoclonal B cell populations. Using proteomic analysis of affinity-purified FBXO45-immunocomplexes and differential whole proteome analysis from GCB-cells of Fbxo45 wt/wt vs Fbxo45 fl/fl mice, we discovered that FBXO45 targets the RHO guanine exchange factor GEF-H1 for ubiquitin-mediated proteasomal degradation. FBXO45 exclusively interacts with GEF H1 among 8 F-box proteins investigated and silencing of FBXO45 using three independent shRNA and CRISPR-Cas9-mediated knockouts in B-cell lymphoma cell lines promotes RHOA and MAPK activation, B cell growth and enhances proliferation. GEF-H1 is stabilized by FBXO45 depletion and GEF-H1 ubiquitination by FBXO45 requires phosphorylation of GEF-H1. Importantly, FBXO45 depletion and expression of a GEF-H1 mutant that is unable to bind FBXO45 results in GEF-H1 stabilization, promotes hyperactivated RHO and MAPK signaling and B-cell oncogenicity in vitro and in vivo. Notably, this phenotype is reverted by co-silencing of GEF-H1. Inducible ectopic expression of FBXO45 triggers accelerated turnover of GEF H1 and decreased RHOA signaling. Genomic analyses revealed recurrent loss targeting FBXO45 in transformed DLBCL (25%), de novo DLBCL (6.6%) and FL (2.3%). In keeping with our observation of prolonged hyperactivation of pERK1/2 consequent to FBXO45 ablation, in vitro and in vivo studies using B-cell lymphoma cell lines and xenografts demonstrated increased sensitivity to pharmacologic blockade with the MAP2K1/2 (ERK1/2) inhibitor Trametinib. Conclusions: Our findings define a novel FBXO45-GEF-H1-MAPK signalling axis, which plays an important role in DLBCL pathogenesis. Our studies carry implications for potential exploitation of this pathway for targeted therapies. Disclosures Siebert: AstraZeneca: Speakers Bureau. Lim: EUSA Pharma: Honoraria.

An antibody-drug conjugate directed to the ALK receptor demonstrates efficacy in preclinical models of neuroblastoma

2019 ◽  
Vol 11 (483) ◽  
pp. eaau9732 ◽  
Author(s):  
Renata Sano ◽  
Kateryna Krytska ◽  
Colleen E. Larmour ◽  
Pichai Raman ◽  
Daniel Martinez ◽  
...  
Keyword(s):  
Cell Surface ◽  
Neuroblastoma Cells ◽  
In Vivo Studies ◽  
Wild Type ◽  
Antibody Drug Conjugate ◽  
Cell Surface Antigens ◽  
Drug Conjugates ◽  
Bona Fide ◽  
Antibody Drug

Enthusiasm for the use of antibody-drug conjugates (ADCs) in cancer therapy has risen over the past few years. The success of this therapeutic approach relies on the identification of cell surface antigens that are widely and selectively expressed on tumor cells. Studies have shown that native ALK protein is expressed on the surface of most neuroblastoma cells, providing an opportunity for development of immune-targeting strategies. Clinically relevant antibodies for this target have not yet been developed. Here, we describe the development of an ALK-ADC, CDX-0125-TEI, which selectively targets both wild-type and mutated ALK-expressing neuroblastomas. CDX-0125-TEI exhibited efficient antigen binding and internalization, and cytotoxicity at picomolar concentrations in cells with different expression of ALK on the cell surface. In vivo studies showed that CDX-0125-TEI is effective against ALK wild-type and mutant patient-derived xenograft models. These data demonstrate that ALK is a bona fide immunotherapeutic target and provide a rationale for clinical development of an ALK-ADC approach for neuroblastomas and other ALK-expressing childhood cancers such as rhabdomyosarcomas.

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