scholarly journals Reconstitution of Modified Chromatin Templates for In Vitro Functional Assays

2011 ◽  
pp. 237-253 ◽  
Author(s):  
Miyong Yun ◽  
Chun Ruan ◽  
Jae-Wan Huh ◽  
Bing Li
Keyword(s):  
Functional Assays

scholarly journals Evaluation of the Expression of CCR5 and CX3CR1 Receptors and Correlation with the Functionality of T Cells in Women infected with ZIKV during Pregnancy

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 191
Author(s):  
Débora Familiar-Macedo ◽  
Iury Amancio Paiva ◽  
Jessica Badolato-Corrêa da Silva ◽  
Fabiana Rabe de Carvalho ◽  
Helver Gonçalves Dias ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Clinical Outcome ◽  
Cell Responses ◽  
Functional Assays ◽  
Asymptomatic Children ◽  
Cd107a Expression ◽  
Congenital Zika Syndrome ◽  
Ifn Γ

There have been reports of neurological abnormalities associated with the Zika virus (ZIKV), such as congenital Zika syndrome (CZS) in children born to mothers infected during pregnancy. We investigated how the immune response to ZIKV during pregnancy is primed and conduct a thorough evaluation of the inflammatory and cytotoxic profiles as well as the expression of CCR5 and CX3CR1. We compared the reactivity of T cells to ZIKV peptides in convalescent mothers infected during pregnancy. The child’s clinical outcome (i.e., born with or without CZS) was taken to be the variable. The cells were stimulated in vitro with ZIKV peptides and evaluated using the ELISPOT and flow cytometry assays. After in vitro stimulation with ZIKV peptides, we observed a tendency toward a higher Interferon gamma (IFN-γ)-producing T cell responses in mothers who had asymptomatic children and a higher CD107a expression in T cells in mothers who had children with CZS. We found a higher frequency of T cells expressing CD107a+ and co-expressing CX3CR1+CCR5+, which is much clearer in the T cells of mothers who had CZS children. We suggest that this differential profile influenced the clinical outcome of babies. These data need to be further investigated, including the evaluation of other ZIKV peptides and markers and functional assays.

scholarly journals A Novel Human Ada2 Homologue Functions with Gcn5 or Brg1 To Coactivate Transcription

2003 ◽  
Vol 23 (19) ◽  
pp. 6944-6957 ◽  
Author(s):  
Nickolai A. Barlev ◽  
Alexander V. Emelyanov ◽  
Paola Castagnino ◽  
Philip Zegerman ◽  
Andrew J. Bannister ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Chromatin Remodeling ◽  
Adaptor Protein ◽  
Saga Complex ◽  
Yeast Two Hybrid ◽  
Functional Assays ◽  
Two Hybrid ◽  
Histone Acetyltransferase Activity

ABSTRACT In yeast, the transcriptional adaptor yeast Ada2 (yAda2) is a part of the multicomponent SAGA complex, which possesses histone acetyltransferase activity through action of the yGcn5 catalytic enzyme. yAda2, among several SAGA proteins, serves to recruit SAGA to genes via interactions with promoter-bound transcription factors. Here we report identification of a new human Ada2 homologue, hAda2β. Ada2β differs both biochemically and functionally from the previously characterized hAda2α, which is a stable component of the human PCAF (human Gcn5 homologue) acetylase complex. Ada2β, relative to Ada2α, interacted selectively, although not stably, with the Gcn5-containing histone acetylation complex TFTC/STAGA. In addition, Ada2β interacted with Baf57 (a component of the human Swi/Snf complex) in a yeast two-hybrid screen and associated with human Swi/Snf in vitro. In functional assays, hAda2β (but not Ada2α), working in concert with Gcn5 (but not PCAF) or Brg1 (the catalytic component of hSwi/Snf complex), increased transcription via the B-cell-specific transcription factor Pax5/BSAP. These findings support the view that Gcn5 and PCAF have distinct roles in vivo and suggest a new mechanism of coactivator function, in which a single adaptor protein (Ada2β) can coordinate targeting of both histone acetylation and chromatin remodeling activities.

scholarly journals REGULATION OF THE IMMUNE RESPONSE

1973 ◽  
Vol 137 (3) ◽  
pp. 721-739 ◽  
Author(s):  
Michael Hoffmann ◽  
John W. Kappler
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Immune Suppression ◽  
Antigen Recognition ◽  
Helper T Cells ◽  
Antibody Specificity ◽  
Humoral Immune ◽  
Functional Assays ◽  
Humoral Immune Responses

The specificity of antigen recognition by thymus-derived helper cells (T cells) and antibody was examined in mice, heterologous erythrocyte antigens from sheep (SRBC), goat (GRBC), burro (BRBC), chicken (CRBC), and toad (TRBC) being used. Antibody specificity was tested by a number of functional assays: hemagglutination, hemolysis, and immune suppression. The specificity of T cells was determined by titrating their ability to help the in vitro antitrinitrophenol (TNP) responses of mouse spleen cultures immunized with the hapten coupled to the various test erythrocytes as carrier. Anti-SRBC antibody cross-reacted with GRBC, but not with BRBC, CRBC, or TRBC. In contrast, SRBC-primed helper T cells cross-reacted with both GRBC and BRBC, but not with CRBC or TRBC, indicating a difference in the specificity of antigen recognition between the cellular and the humoral immune responses.

scholarly journals PKAc-directed interaction and phosphorylation of Ptc is required for Hh signaling inhibition in Drosophila

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Jialin Fan ◽  
Yajie Gao ◽  
Yi Lu ◽  
Wenqing Wu ◽  
Shuo Yuan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Kinase Activity ◽  
Functional Assays ◽  
Working Model ◽  
Inhibitory Function ◽  
Hh Signaling ◽  
Signaling Activation

Abstract Ptc is a gatekeeper to avoid abnormal Hh signaling activation, but the key regulators involved in Ptc-mediated inhibition remain largely unknown. Here, we identify PKAc as a key regulator required for Ptc inhibitory function. In the absence of Hh, PKAc physically interacts with Ptc and phosphorylates Ptc at Ser-1150 and -1183 residues. The presence of Hh unleashes PKAc from Ptc and activates Hh signaling. By combining both in vitro and in vivo functional assays, we demonstrate that such Ptc–PKAc interaction and Ptc phosphorylation are both important for Ptc inhibitory function. Interestingly, we further demonstrate that PKAc is subjected to palmitoylation, contributing to its kinase activity on plasma membrane. Based on those novel findings, we establish a working model on Ptc inhibitory function: In the absence of Hh, PKAc interacts with and phosphorylates Ptc to ensure its inhibitory function; and Hh presence releases PKAc from Ptc, resulting in Hh signaling activation.

Comparison between Progenitor Cells from Mobilized Peripheral Blood (PB) in Healthy Donors and Non-Hodgkin’s Lymphoma (NHL) Patients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5002-5002
Author(s):  
Eva M. Villaron ◽  
Julia Almeida ◽  
Natalia Lopez-Holgado ◽  
Fermin M. Sanchez-Guijo ◽  
Mercedes Alberca ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
Functional Assays ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
Cell Subpopulations ◽  
The Impact ◽  
Pbsc Mobilization

Abstract INTRODUCTION: Peripheral blood stem cell (PBSC) mobilization is impaired in patients receiving chemotherapy but, as far as we know there is no data about the impact of chemotherapy on different PB progenitor cell subpopulations. AIM: to ascertain whether or not immature or committed progenitor cell are affected by chemotherapy prior PBSC mobilization in NHL patients. MATERIAL AND METHODS: a total of 27 PB samples from NHL patients and 36 PB samples from healthy donors were studied. Immunophenotypic analysis of CD34+ cell subpopulations was performed using the following four colour combinations of monoclonal antibodies (FITC/PE/PC5/APC): CD90/CD133/CD38/CD34 and CD71/CD13/CD45/CD34. In order to study committed progenitor cells “in vitro”, standard colony-forming assays were used and, in order to investigate the behaviour of the uncommitted progenitors Delta Assays of plastic adherent progenitor cells (PΔ) were performed. RESULTS: The comparison between NHL patients and healthy donors is shown in Table 1. The relationship between data obtained by flow cytometry and cultures was statistically significant (p<0.05, r>0.568) for all the progenitors analysed. Table 1: Results of Immunophenotypic and Functional Assays LNH patients Healthy donors p Data expressed as median (range). 1. Percentage among CD34+ cells. 2. Number of CFU/10 5 planted cells. 3. Number of CFU/10 6 planted cells % CD34 0.16(0.04–3.65) 0.57(0.11–1.81) 0.013 Immunophenotypic Data Erithroid 1 0.05(0.01–0.60) 0.14(0.02–0.42) 0.098 Myelo–monocytic 1 0.11(0.02–2.41) 0.37(0.07–1.18) 0.014 Immature 1 0.01(0.00–0.63) 0.05(0.01–0.19) 0.014 CFU-GM 2 70(4–440) 90(0–904) 0.327 Clonogenic and Delta Assays data BFU-E 2 62(6–172) 85(0–240) 0.046 CFU-Mix 2 18(0–124) 42(0–140) 0.018 CFU Δ3 356(0–3509) 953(90–8320) 0.033 CONCLUSIONS: We can conclude that in NHL, mobilized committed and above all immature progenitors are impaired when compared with healthy subjects, both analysed by immunological and functional assays. Only granulomonocytic progenitors analysed by a functional approach seemed to be preserved.

Higher plant microtubule-associated proteins: In vitro functional assays

Biochimie ◽  
1993 ◽  
Vol 75 (8) ◽  
pp. 725-730 ◽  
Author(s):  
M. Vantard ◽  
P. Schellenbaum ◽  
C. Peter ◽  
A.M. Lambert
Keyword(s):  
Higher Plant ◽  
Microtubule Associated Proteins ◽  
Functional Assays ◽  
Associated Proteins

scholarly journals Specific binding of leukotriene B4 to a receptor on human polymorphonuclear leukocytes.

1983 ◽  
Vol 157 (2) ◽  
pp. 628-641 ◽  
Author(s):  
R A Kreisle ◽  
C W Parker
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Specific Binding ◽  
Leukotriene B4 ◽  
Trans Isomers ◽  
Functional Assays ◽  
Human Polymorphonuclear Leukocytes ◽  
Fold Excess

In this paper we have described the binding of nanomoler concentrations of [3H]leukotriene B4 (LTB4) to human polymorphonuclear leukocytes. Because up to 80% of the total [3H]LTB4 binding was blocked by excess (greater than 100 times) [14C]LTB4, the majority of binding is specific. Stereospecificity of the LTB4 binding is demonstrated by the diminished relative abilities of the 6-trans-and 12-epi-6-trans- isomers of LTB4 to block [3H]LTB4 binding. With these two isomers 3-10-fold higher than [14C]LTB4 concentrations were needed for equivalent inhibition of [3H]LTB4 binding. This difference is quantitatively less dramatic than the differences between these isomers in many in vitro functional assays such as chemokinesis, chemotaxis, and degranulation. Binding of [3H]FMLP is not blocked at greater than 100-fold excess of LTB4. The binding of [3H]LTB4 to cells appears to be essentially irreversible at 4 degrees C, but not at 37 degrees C where initially bound LTB4 is rapidly converted to metabolites which then enter the medium. These results suggest the presence of a saturable, stereospecific site for LTB4 on PMN. The association of LTB4 binding and the initiation of pharmacological responses to LTB4 will require further studies.

scholarly journals The C-Terminal Domain of Cernunnos/XLF Is Dispensable for DNA Repair In Vivo

2008 ◽  
Vol 29 (5) ◽  
pp. 1116-1122 ◽  
Author(s):  
Laurent Malivert ◽  
Isabelle Callebaut ◽  
Paola Rivera-Munoz ◽  
Alain Fischer ◽  
Jean-Paul Mornon ◽  
...  
Keyword(s):  
Dna Repair ◽  
Nonhomologous End Joining ◽  
Dna Ligase ◽  
Repair Pathway ◽  
End Joining ◽  
Functional Assays ◽  
The Core ◽  
Ligase Iv

ABSTRACT The core nonhomologous end-joining DNA repair pathway is composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos). Although Cernunnos and X4 are structurally related and participate in the same complex together with L4, they have distinct functions during DNA repair. L4 relies on X4 but not on Cernunnos for its stability, and L4 is required for optimal interaction of Cernunnos with X4. We demonstrate here, using in vitro-generated Cernunnos mutants and a series of functional assays in vivo, that the C-terminal region of Cernunnos is dispensable for its activity during DNA repair.

scholarly journals Fast and Efficient Genome Editing of Human FOXP3+ Regulatory T Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Lauren Van Zeebroeck ◽  
Rebeca Arroyo Hornero ◽  
Beatriz F. Côrte-Real ◽  
Ibrahim Hamad ◽  
Torsten B. Meissner ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Genome Editing ◽  
Therapeutic Target ◽  
Peripheral Tolerance ◽  
Immune Homeostasis ◽  
Cell Integrity ◽  
Potential Therapeutic Target ◽  
Functional Assays

FOXP3+ regulatory T cells (Tregs) are central for maintaining peripheral tolerance and immune homeostasis. Because of their immunosuppressive characteristics, Tregs are a potential therapeutic target in various diseases such as autoimmunity, transplantation and infectious diseases like COVID-19. Numerous studies are currently exploring the potential of adoptive Treg therapy in different disease settings and novel genome editing techniques like CRISPR/Cas will likely widen possibilities to strengthen its efficacy. However, robust and expeditious protocols for genome editing of human Tregs are limited. Here, we describe a rapid and effective protocol for reaching high genome editing efficiencies in human Tregs without compromising cell integrity, suitable for potential therapeutic applications. By deletion of IL2RA encoding for IL-2 receptor α-chain (CD25) in Tregs, we demonstrated the applicability of the method for downstream functional assays and highlighted the importance for CD25 for in vitro suppressive function of human Tregs. Moreover, deletion of IL6RA (CD126) in human Tregs elicits cytokine unresponsiveness and thus may prevent IL-6-mediated instability of Tregs, making it an attractive target to potentially boost functionality in settings of adoptive Treg therapies to contain overreaching inflammation or autoimmunity. Thus, our rapid and efficient protocol for genome editing in human Tregs may advance possibilities for Treg-based cellular therapies.

scholarly journals Functional Mapping of Adhesiveness on Live Cells Reveals How Guidance Phenotypes Can Emerge From Complex Spatiotemporal Integrin Regulation

2021 ◽  
Vol 9 ◽  
Author(s):  
Philippe Robert ◽  
Martine Biarnes-Pelicot ◽  
Nicolas Garcia-Seyda ◽  
Petra Hatoum ◽  
Dominique Touchard ◽  
...  
Keyword(s):  
Distance Effect ◽  
Cell Polarization ◽  
Functional Mapping ◽  
Live Cells ◽  
Long Distance ◽  
Adhesion Properties ◽  
Functional Assays ◽  
New Perspective

Immune cells have the ubiquitous capability to migrate disregarding the adhesion properties of the environment, which requires a versatile adaptation of their adhesiveness mediated by integrins, a family of specialized adhesion proteins. Each subtype of integrins has several ligands and several affinity states controlled by internal and external stimuli. However, probing cell adhesion properties on live cells without perturbing cell motility is highly challenging, especially in vivo. Here, we developed a novel in vitro method using micron-size beads pulled by flow to functionally probe the local surface adhesiveness of live and motile cells. This method allowed a functional mapping of the adhesiveness mediated by VLA-4 and LFA-1 integrins on the trailing and leading edges of live human T lymphocytes. We show that cell polarization processes enhance integrin-mediated adhesiveness toward cell rear for VLA-4 and cell front for LFA-1. Furthermore, an inhibiting crosstalk of LFA-1 toward VLA-4 and an activating crosstalk of VLA-4 toward LFA-1 were found to modulate cell adhesiveness with a long-distance effect across the cell. These combined signaling processes directly support the bistable model that explains the emergence of the versatile guidance of lymphocyte under flow. Molecularly, Sharpin, an LFA-1 inhibitor in lymphocyte uropod, was found involved in the LFA-1 deadhesion of lymphocytes; however, both Sharpin and Myosin inhibition had a rather modest impact on adhesiveness. Quantitative 3D immunostaining identified high-affinity LFA-1 and VLA-4 densities at around 50 and 100 molecules/μm2 in basal adherent zones, respectively. Interestingly, a latent adhesiveness of dorsal zones was not grasped by immunostaining but assessed by direct functional assays with beads. The combination of live functional assays, molecular imaging, and genome editing is instrumental to characterizing the spatiotemporal regulation of integrin-mediated adhesiveness at molecular and cell scales, which opens a new perspective to decipher sophisticated phenotypes of motility and guidance.

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