scholarly journals One-Step Enzymatic Modification of the Cell Surface Redirects Cellular Cytotoxicity and Parasite Tropism

2014 ◽  
Vol 10 (2) ◽  
pp. 460-465 ◽  
Author(s):  
Lee Kim Swee ◽  
Sebastian Lourido ◽  
George W. Bell ◽  
Jessica R. Ingram ◽  
Hidde L. Ploegh
Keyword(s):  
Cell Surface ◽  
Cellular Cytotoxicity ◽  
Enzymatic Modification ◽  
One Step

Protein cell-surface display through in situ enzymatic modification of proteins with a poly(Ethylene glycol)-lipid

2013 ◽  
Vol 110 (10) ◽  
pp. 2785-2789 ◽  
Author(s):  
Urara Tomita ◽  
Satoshi Yamaguchi ◽  
Yasukazu Maeda ◽  
Kazuki Chujo ◽  
Kosuke Minamihata ◽  
...  
Keyword(s):  
Cell Surface ◽  
Ethylene Glycol ◽  
Surface Display ◽  
Cell Surface Display ◽  
Enzymatic Modification ◽  
Poly Ethylene Glycol ◽  
Poly Ethylene ◽  
Protein Cell

scholarly journals Involvement of a cell surface protein and an ecto-protein kinase in myogenesis

1991 ◽  
Vol 279 (2) ◽  
pp. 475-482 ◽  
Author(s):  
X Y Chen ◽  
T C Y Lo
Keyword(s):  
Protein Kinase ◽  
Cell Surface ◽  
Myogenic Differentiation ◽  
Morphological Differentiation ◽  
Surface Protein ◽  
Chemical Reagents ◽  
Defective Mutant ◽  
A Cell ◽  
One Step ◽  
Multinucleated Myotubes

Myogenic differentiation is composed of a sequential cascade of multiple steps leading to the formation of multinucleated myotubes. The interference with any one step would abolish myogenesis. The present investigation examined the cell surface components which might be involved in myogenesis. Studies with subconfluent day 2 cultures of rat L6 myoblasts revealed that a cell surface 112 kDa protein was phosphorylated by a Ca(2+)-, F(-)- and Mg(2+)-dependent ecto-protein kinase [Chen & Lo (1991) Biochem. J. 279, 467-474]. We have shown in the present investigation that adequate ATP was present on the cell surface for efficient functioning of this ecto-protein kinase. The phosphorylation of the 112 kDa protein by this ecto-protein kinase was decrease dramatically in confluent cells and in multinucleated myotubes. The following evidence suggests that both the 112 kDa protein and the ecto-protein kinase may play important roles in myogenesis. (i) The highest phosphorylation activity was observed in subconfluent cultures, i.e. before the onset of morphological differentiation. (ii) Treatment of cells with chemical reagents resulted in a corresponding decrease in the ecto-protein kinase, the 112 kDa protein, the phosphorylated 112 kDa protein (p112) and the ability to form myotubes. (iii) The level of p112 in a conditional myogenesis-defective mutant corresponded with the cells' eventual ability to differentiate. (iv) A mutant defective in the ecto-protein kinase was impaired in the phosphorylation of the 112 kDa protein and in myogenesis. (v) A mutant containing only residual levels of the 112 kDa protein was deficient in both p112 and myogenesis. (vi) Since the level of p112 was normal in another myogenesis-defective mutant, the phosphorylation of this protein was not likely to be a consequence of myogenic differentiation. The above findings suggest that the ecto-protein kinase and the 112 kDa protein may directly or indirectly be associated with the myogenic pathway. Since the levels of the ecto-protein kinase, the 112 kDa protein and p112 decreased dramatically upon the formation of myotubes, these proteins were probably not required once morphological differentiation had been initiated.

scholarly journals Direct dilution of cell aliquot for high temporal resolution bacterial surface charge measurement

2015 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Cell Surface ◽  
Sample Preparation ◽  
Surface Charge ◽  
Temporal Resolution ◽  
Gold Standard ◽  
Preparation Technique ◽  
Dilution Factor ◽  
Error Type ◽  
Bacterial Surface ◽  
One Step

Bacterial surface charge (SC) mediates important cell-environment and microbe-host interactions, and its accurate and precise measurement by microelectrophoresis requires removing metabolites adhered to the cell surface - where repeated centrifugation and washing by buffers is the gold standard method. Unfortunately, the need for time-consuming centrifugation limits the temporal resolution of sampling and interrogation of experimental dynamics; especially for samples requiring immediate treatment post sampling. Herein, the feasibility of diluting cell aliquots with buffer as a one-step sample preparation technique for SC measurement was investigated by characterising the effects of dilution factor, type of cation, and buffer conductivity on measuring SC of Escherichia coli DH5α grown in LB medium. Results indicated that dilution factor was critical to accurate SC measurement since low signal-to-noise ratios in high or low cell concentration samples generated substantial error. Type of buffer cation was also important since putative binding of high affinity cations to the cell surface underestimated SC of negatively-charged bacteria. Finally, although high conductivity buffers enabled greater removal of adsorbed metabolites through increased charge screening, a broader statistical distribution of measured SC was also observed – which, at extreme conductivity values, led to inaccurate data, probably due to removal of both intrinsic cell surface ions and exogenous adsorbed metabolites. Altogether, one-step dilution of cell aliquot with deionized water reliably reproduced E. coli SC values obtained via the gold standard approach; however, since the ensemble of secreted metabolites is bacteria/medium specific, distinct diluent and optimal parameters exist for each system. The described methodology may find use in preparing samples for cell surface characterisation studies, where it would help reduce sample preparation time – and thus, improve temporal resolution at which scientific questions can be probed and answered.

scholarly journals Direct dilution of cell aliquot for high temporal resolution bacterial surface charge measurement

2016 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Cell Surface ◽  
Sample Preparation ◽  
Surface Charge ◽  
Temporal Resolution ◽  
Gold Standard ◽  
Preparation Technique ◽  
Dilution Ratio ◽  
Bacterial Surface ◽  
Charge Measurement ◽  
One Step

Bacterial surface charge mediates important cell-environment and microbe-host interactions, and its accurate and precise measurement by microelectrophoresis requires removing metabolites adhered to the cell surface - where repeated centrifugation and washing by buffers is the gold standard sample preparation method. Unfortunately, the need for time-consuming centrifugation limits the temporal resolution of sampling and profiling of experimental dynamics; especially for samples requiring immediate treatment after sampling. Herein, the feasibility of diluting cell aliquots with buffer as a one step sample preparation technique for surface charge measurement was investigated by characterizing the effects of dilution ratio, cation type, and buffer conductivity on measuring surface charge of Escherichia coli DH5α (ATCC 53868) grown in LB Lennox medium. Results indicated that dilution ratio was critical to accurate surface charge measurement since low signal-to-noise ratio in high or low cell concentration samples generated substantial error. Type of buffer cation was also important since putative binding of high affinity cations to the cell surface underestimated surface charge of negatively charged bacteria. Finally, high conductivity buffers enabled greater removal of adsorbed metabolites through increased charge screening. However, a broader statistical distribution of measured surface charge was also observed – which, at extreme conductivity values, led to inaccurate data; probably due to removal of both intrinsic cell surface ions and exogenous adsorbed metabolites. Altogether, one step dilution of cell aliquot with deionized water reliably reproduced E. coli surface charge values obtained via the gold standard approach. But, since the ensemble of secreted metabolites is bacteria and/or medium specific, distinct diluent and experiment parameters exist for each system. The described methodology may find use in preparing samples for cell surface characterization studies, where it would help reduce sample preparation time – and thus, improve temporal resolution at which scientific questions can be probed and answered.

scholarly journals Incomplete Downregulation of CD4 Expression Affects HIV-1 Env Conformation and Antibody-Dependent Cellular Cytotoxicity Responses

2018 ◽  
Vol 92 (13) ◽  
pp. e00484-18 ◽  
Author(s):  
Jérémie Prévost ◽  
Jonathan Richard ◽  
Halima Medjahed ◽  
Audrey Alexander ◽  
Jennifer Jones ◽  
...  
Keyword(s):  
Cell Surface ◽  
Reporter Gene ◽  
Large Scale ◽  
Surface Expression ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Content Type ◽  
Infected Cells ◽  
Infectious Molecular Clones ◽  
Hiv 1

ABSTRACTHIV-1-infected cells expressing envelope glycoproteins (Env) in the CD4-bound conformation on their surfaces are targeted by antibody-dependent cellular cytotoxicity (ADCC) mediated by CD4-induced (CD4i) antibodies and sera from HIV-1-infected individuals (HIV+sera). By downregulating the surface expression of CD4, Nef prevents Env-CD4 interaction, thus protecting HIV-1-infected cells from ADCC. HIV-1 infectious molecular clones (IMCs) are widely used to measure ADCC. In order to facilitate the identification of infected cells and high-throughput ADCC analysis, reporter genes (e.g., theRenillaluciferase [LucR] gene) are often introduced into IMC constructs. We evaluated the susceptibility of HIV-1-infected CD4+T lymphocytes to ADCC using a panel of parental IMCs and derivatives that expressed the LucR reporter gene, utilizing different molecular strategies, including one specifically designed to retain Nef expression. We found that in some of these constructs, Nef expression in CD4+T cells was suboptimal, and consequently, CD4 downregulation was incomplete. CD4 molecules remaining on the cell surface resulted in the exposure of ADCC-mediating CD4i epitopes on Env and a dramatic increase in the susceptibility of the infected cells to ADCC. Strikingly, protection from ADCC was observed when cells were infected with the parental IMC, which exhibited strong CD4 downregulation. This discrepancy between the parental and Nef-impaired viruses was independent of the strains of Env expressed, but rather, it was correlated with the levels of CD4 surface expression. Overall, our results indicate that caution should be taken when selecting IMCs for ADCC measurements and that CD4 downregulation needs to be carefully monitored when drawing conclusions about the nature and magnitude of ADCC.IMPORTANCEIn-depth understanding of the susceptibility of HIV-1-infected cells to ADCC might help establish correlates of vaccine protection and guide the development of HIV-1 vaccine strategies. Different ADCC assays have been developed, including those using infectious molecular clones (IMCs) carrying a LucR reporter gene that greatly facilitates large-scale quantitative analysis. We previously reported different molecular strategies for introducing LucR while maintaining Nef expression and function and, consequently, CD4 surface downregulation. Here, we demonstrate that utilizing IMCs that exhibit impaired Nef expression can have undesirable consequences due to incomplete CD4 downregulation. CD4 molecules remaining on the cell surface resulted in the exposure of ADCC-mediating CD4i epitopes on Env and a dramatic increase in the susceptibility of the infected cells to ADCC. Overall, our results indicate that CD4 downregulation needs to be carefully monitored when drawing conclusions about the nature and magnitude of ADCC.

scholarly journals Cell Surface Antibody Retention Influences In Vivo Antitumor Activity Mediated by Antibody-dependent Cellular Cytotoxicity

2016 ◽  
Vol 36 (11) ◽  
pp. 5937-5944
Author(s):  
MASAHIRO IKEDA ◽  
KAZUNORI KATO ◽  
MIKI YAMAGUCHI ◽  
HIROFUMI HAMADA ◽  
KAZUYASU NAKAMURA ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Cell Surface ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
In Vivo Antitumor Activity ◽  
Surface Antibody

scholarly journals BST-2 Expression Modulates Small CD4-Mimetic Sensitization of HIV-1-Infected Cells to Antibody-Dependent Cellular Cytotoxicity

2017 ◽  
Vol 91 (11) ◽  
Author(s):  
Jonathan Richard ◽  
Jérémie Prévost ◽  
Benjamin von Bredow ◽  
Shilei Ding ◽  
Nathalie Brassard ◽  
...  
Keyword(s):  
Cell Surface ◽  
Immune Evasion ◽  
Surface Expression ◽  
Hiv Positive ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT Antibodies recognizing conserved CD4-induced (CD4i) epitopes on human immunodeficiency virus type 1 (HIV-1) Env and able to mediate antibody-dependent cellular cytotoxicity (ADCC) have been shown to be present in sera from most HIV-1-infected individuals. These antibodies preferentially recognize Env in its CD4-bound conformation. CD4 downregulation by Nef and Vpu dramatically reduces exposure of CD4i HIV-1 Env epitopes and therefore reduce the susceptibility of HIV-1-infected cells to ADCC mediated by HIV-positive (HIV+) sera. Importantly, this mechanism of immune evasion can be circumvented with small-molecule CD4 mimetics (CD4mc) that are able to transition Env into the CD4-bound conformation and sensitize HIV-1-infected cells to ADCC mediated by HIV+ sera. However, HIV-1 developed additional mechanisms to avoid ADCC, including Vpu-mediated BST-2 antagonism, which decreases the overall amount of Env present at the cell surface. Accordingly, BST-2 upregulation in response to alpha interferon (IFN-α) was shown to increase the susceptibility of HIV-1-infected cells to ADCC despite the activity of Vpu. Here we show that BST-2 upregulation by IFN-β and interleukin-27 (IL-27) also increases the surface expression of Env and thus boosts the ability of CD4mc to sensitize HIV-1-infected cells to ADCC by sera from HIV-1-infected individuals. IMPORTANCE HIV-1 evolved sophisticated strategies to conceal Env epitopes from ADCC-mediating antibodies present in HIV+ sera. Vpu-mediated BST-2 downregulation was shown to decrease ADCC responses by limiting the amount of Env present at the cell surface. This effect of Vpu was shown to be attenuated by IFN-α treatment. Here we show that in addition to IFN-α, IFN-β and IL-27 also affect Vpu-mediated BST-2 downregulation and greatly enhance ADCC responses against HIV-1-infected cells in the presence of CD4mc. These findings may inform strategies aimed at HIV prevention and eradication.

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