scholarly journals Development of Nanobodies as first-in-class inhibitors for the NEDP1 deNEDDylating enzyme

2020 ◽  
Author(s):  
Naima Abidi ◽  
Helene Trauchessec ◽  
Gholamreza Hassanzadeh-Ghassabeh ◽  
Martine Pugniere ◽  
Serge Muyldermans ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Therapeutic Intervention ◽  
Post Translational Modification ◽  
High Affinity ◽  
Culture Cells ◽  
Cell Extracts ◽  
Tissue Culture Cells ◽  
Biochemical Assays ◽  
Processing Activity

AbstractProtein NEDDylation emerges as an important post-translational modification and an attractive target for therapeutic intervention. Modification of NEDD8 onto substrates is finely balanced by the co-ordinated activity of conjugating and deconjugating enzymes. The NEDP1/DEN1/SENP8 protease is a NEDD8 specific processing and deconjugating enzyme that regulates the NEDDylation mainly of non-cullin substrates. Here, we report the development and characterisation of nanobodies as first-in-class inhibitors for NEDP1. The nanobodies display high-affinity (low nM) against NEDP1 and specifically inhibit NEDP1 processing activity in vitro and NEDP1 deconjugating activity in tissue-culture cells and in cell extracts. We also isolated nanobodies that bind to NEDP1 with high-affinity but do not affect NEDP1 activity. The developed nanobodies provide new tools to study the function of NEDP1 and to prevent deNEDDylation in cell extracts used in biochemical assays.

scholarly journals The glycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway.

1990 ◽  
Vol 110 (3) ◽  
pp. 637-649 ◽  
Author(s):  
K G Rothberg ◽  
Y S Ying ◽  
J F Kolhouse ◽  
B A Kamen ◽  
R G Anderson
Keyword(s):  
Tissue Culture ◽  
Folate Receptor ◽  
Endocytic Pathway ◽  
Coated Pits ◽  
Folate Binding Protein ◽  
Glycosyl Phosphatidylinositol ◽  
High Affinity ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Endocytic Machinery

The folate receptor, also known as the membrane folate-binding protein, is maximally expressed on the surface of folate-depleted tissue culture cells and mediates the high affinity accumulation of 5-methyltetrahydrofolic acid in the cytoplasm of these cells. Recent evidence suggests that this receptor recycles during folate internalization and that it is anchored in the membrane by a glycosyl-phosphatidylinositol linkage. Using quantitative immunocytochemistry, we now show that (a) this receptor is highly clustered on the cell surface; (b) these clusters are preferentially associated with uncoated membrane invaginations rather than clathrin-coated pits; and (c) the receptor is not present in endosomes or lysosomes. This receptor appears to physically move in and out of the cell using a novel uncoated pit pathway that does not merge with the clathrin-coated pit endocytic machinery.

scholarly journals Cultivation of Pathogenic and Opportunistic Free-Living Amebas

2002 ◽  
Vol 15 (3) ◽  
pp. 342-354 ◽  
Author(s):  
Frederick L. Schuster
Keyword(s):  
Tissue Culture ◽  
Clinical Samples ◽  
Human Pathogens ◽  
Free Living ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Feeder Layers ◽  
Important Means ◽  
Human Infections

SUMMARY Free-living amebas are widely distributed in soil and water, particularly members of the genera Acanthamoeba and Naegleria. Since the early 1960s, they have been recognized as opportunistic human pathogens, capable of causing infections of the central nervous system (CNS) in both immunocompetent and immunocompromised hosts. Naegleria is the causal agent of a fulminant CNS condition, primary amebic meningoencephalitis; Acanthamoeba is responsible for a more chronic and insidious infection of the CNS termed granulomatous amebic encephalitis, as well as amebic keratitis. Balamuthia sp. has been recognized in the past decade as another ameba implicated in CNS infections. Cultivation of these organisms in vitro provides the basis for a better understanding of the biology of these amebas, as well as an important means of isolating and identifying them from clinical samples. Naegleria and Acanthamoeba can be cultured axenically in cell-free media or on tissue culture cells as feeder layers and in cultures with bacteria as a food source. Balamuthia, which has yet to be isolated from the environment, will not grow on bacteria. Instead, it requires tissue culture cells as feeder layers or an enriched cell-free medium. The recent identification of another ameba, Sappinia diploidea, suggests that other free-living forms may also be involved as causal agents of human infections.

scholarly journals REPAIR OF UV-INDUCED PYRIMIDINE DIMERS IN DROSOPHILA MELANOGASTER CELLS IN VITRO

Genetics ◽  
1973 ◽  
Vol 73 (2) ◽  
pp. 297-302
Author(s):  
James E Trosko ◽  
Kathy Wilder
Keyword(s):  
Tissue Culture ◽  
Drosophila Melanogaster ◽  
Ultraviolet Light ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Pyrimidine Dimers ◽  
Drosophila Cells

ABSTRACT Tissue culture cells of Drosophila melanogaster were given various doses of ultraviolet light. The results indicate that Drosophila cells do have a darkrepair excision mechanism which is not sensitive to caffeine. Pyrimidine dimers were destroyed by photoreactivating illumination in these cells and this destruction probably represents monomerization of the pyrimidine dimers.

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