scholarly journals Positive and Negative TAFII Functions That Suggest a Dynamic TFIID Structure and Elicit Synergy with TRAPs in Activator-Induced Transcription

2001 ◽  
Vol 21 (20) ◽  
pp. 6882-6894 ◽  
Author(s):  
Mohamed Guermah ◽  
Yong Tao ◽  
Robert G. Roeder
Keyword(s):  
Basal Transcription ◽  
Absolute Level ◽  
Western Blots ◽  
Core Promoters ◽  
Activated State ◽  
Cloned Cdna ◽  
Sequence Relationships ◽  
And Function ◽  
Human Transcription Factor

ABSTRACT Human transcription factor TFIID contains the TATA-binding protein (TBP) and several TBP-associated factors (TAFIIs). To elucidate the structural organization and function of TFIID, we expressed and characterized the product of a cloned cDNA encoding human TAFII135 (hTAFII135). Comparative far Western blots have shown that hTAFII135 interacts strongly with hTAFII20, moderately with hTAFII150, and weakly with hTAFII43 and hTAFII250. Consistent with these observations and with sequence relationships of hTAFII20 and hTAFII135 to histones H2B and H2A, respectively, TFIID preparations that contain higher levels of hTAFII135 also contain higher levels of hTAFII20, and the interaction between hTAFII20 and hTAFII135 is critical for human TFIID assembly in vitro. From a functional standpoint, hTAFII135 has been found to interact strongly and directly with hTFIIA and (within a complex that also contains hTBP and hTAFII250) to specifically cooperate with TFIIA to relieve TAFII250-mediated repression of TBP binding and function on core promoters. Finally, we report a functional synergism between TAFIIs and the TRAP/Mediator complex in activated transcription, manifested as hTAFII-mediated inhibition of basal transcription and a consequent TRAP requirement for both a high absolute level of activated transcription and a high and more physiological activated/basal transcription ratio. These results suggest a dynamic TFIID structure in which the switch from a basal hTAFII-enhanced repression state to an activator-mediated activated state on a promoter may be mediated in part through activator or coactivator interactions with hTAFII135.

scholarly journals Effects of epigallocatechin gallate on regulatory T cell number and function in obese v. lean volunteers

2010 ◽  
Vol 103 (12) ◽  
pp. 1771-1777 ◽  
Author(s):  
Jung-Mi Yun ◽  
Ishwarlal Jialal ◽  
Sridevi Devaraj
Keyword(s):  
Epigallocatechin Gallate ◽  
Cell Number ◽  
Normal Weight ◽  
Western Blots ◽  
Egcg Treatment ◽  
Hdac Activity ◽  
Obese Subjects ◽  
Specific Regulation ◽  
And Function

Obesity predisposes to an increased incidence of diabetes and CVD. Also, obesity is a pro-inflammatory state. Regulatory T cells (Tregs) are essential negative regulators of inflammation and are down-regulated in pro-inflammatory states. Animal models of obesity are associated with decreased Tregs. The dietary modulation of Tregs could be used as a therapeutic strategy to control inflammation. Epigallocatechin gallate (EGCG) is a potent anti-inflammatory agent and an active ingredient of green tea and is suggested to have a role as a preventive agent in obesity, diabetes and CVD. The role of EGCG in the modulation of Tregs has, however, not been studied. Thus, the aim of the present study was to determine the effect of EGCG on the number and function of Tregs in obese and lean human subjects in vitro, and to delineate its specific regulation mechanisms. Tregs were isolated from normal-weight and obese subjects. Tregs were cultured in the absence or presence of EGCG (20 μm) for 24 h. Foxp3-expressing Tregs were enumerated using flow cytometry. Histone deacetylase (HDAC) activity and nuclear NF-κBp65 level were measured by ELISA and Western blots. Obese subjects had lower Tregs and IL-10 production than lean subjects. EGCG treatment significantly enhanced the number of Foxp3-expressing Tregs and IL-10 production in vitro (P < 0·05) in both groups. Also, EGCG decreased NF-κB activity and increased HDAC activity and HDAC-2 expression in Tregs (P < 0·05) in both groups. Thus, in part, EGCG enhances the functionality of Tregs, i.e. IL-10 production and number by suppressing the NF-κB signalling pathway via inducing epigenetic changes.

scholarly journals The steroid receptor antagonists RU40555 and RU486 activate glucocorticoid receptor translocation and are not excreted by the steroid hormones transporter in L929 cells

2001 ◽  
Vol 169 (2) ◽  
pp. 309-320 ◽  
Author(s):  
CM Pariante ◽  
BD Pearce ◽  
TL Pisell ◽  
C Su ◽  
AH Miller
Keyword(s):  
Glucocorticoid Receptor ◽  
Steroid Hormones ◽  
Fibroblast Cell ◽  
Mouse Fibroblast ◽  
Membrane Transporter ◽  
Methyl Radical ◽  
Western Blots ◽  
L929 Cells ◽  
And Function

RU40555 is a recently available glucocorticoid receptor (GR) antagonist that differs from RU486 by a methyl radical. We have used the mouse fibroblast cell line L929 to study the in vitro effects of RU40555 on GR translocation and function and on the membrane steroid hormones transporter. The results showed that: 1) RU40555 competed for the binding of labelled dexamethasone (Dex) with a K(i) of 2.4 nM; 2) both RU40555 and RU486 were equally potent inhibitors of Dex-induced GR-mediated gene transcription; 3) maximum GR translocation induced by micromolar concentrations of Dex and the GR antagonists was approximately 30-55% loss in the cytoplasmic GR and approximately 40-90% increase in the nuclear GR (assessed by GR immunostaining in cytoplasm and nucleus and western blots of immunoprecipitated GR protein in cytosolic and nuclear fractions) and was similar for the two antagonists; 4) at nanomolar concentrations, RU40555 and RU486 induced more GR translocation than Dex (assessed by [(3)H]Dex binding and western blot of immunoreactive GR in the same cytosolic homogenates); 5) blocking the steroids membrane transporter with verapamil (100 microM) in the presence of Dex (10 nM) increased GR translocation to levels similar to those induced by RU40555 (10 nM) and RU486 (10 nM) alone; 6) verapamil did not affect GR translocation in the presence of RU40555 or RU486. These data demonstrate similar quantitative effects on GR translocation by RU486 and the new GR antagonist, RU40555. Moreover, RU40555, like RU486, is an effective GR antagonist. Finally, there is no evidence that the intracellular concentrations of RU40555 or RU486 are regulated by the steroids membrane transporter in L929 cells.

scholarly journals Prolonged Amphetamine Exposures Increase the Endogenous Human Dopamine Receptors 2 at the Cellular Membrane in Cells Lacking the Dopamine Transporter

2021 ◽  
Vol 15 ◽  
Author(s):  
Vindhya Nawaratne ◽  
Sean P. McLaughlin ◽  
Felix P. Mayer ◽  
Zayna Gichi ◽  
Alyssa Mastriano ◽  
...  
Keyword(s):  
Dopamine Transporter ◽  
Specific Inhibitor ◽  
Cellular Membrane ◽  
Hek293 Cells ◽  
Imaging Data ◽  
Western Blots ◽  
Synaptic Terminals ◽  
And Function ◽  
In Cells

The dopamine 2 receptors (D2R) are G-protein coupled receptors expressed both in pre- and post-synaptic terminals that play an important role in mediating the physiological and behavioral effects of amphetamine (Amph). Previous studies have indicated that the effects of Amph at the D2R mainly rely on the ability of Amph to robustly increase extracellular dopamine through the dopamine transporter (DAT). This implies that the effects of Amph on D2R require the neurotransmitter dopamine. However, because of its lipophilic nature, Amph can cross the cellular membrane and thus potentially affect D2R expression independently of dopamine and DAT, e.g., in post-synaptic terminals. Here we used an in vitro system to study whether Amph affects total expression, cellular distribution, and function of the human D2R (hD2R), endogenously expressed in HEK293 cells. By performing Western blot experiments, we found that prolonged treatments with 1 or 50 μM Amph cause a significant decrease of the endogenous hD2R in cells transfected with human DAT (hDAT). On the other hand, in cells lacking expression of DAT, quantification of the hD2R-mediated changes in cAMP, biotinylation assays, Western blots and imaging experiments demonstrated an increase of hD2R at the cellular membrane after 15-h treatments with Amph. Moreover, imaging data suggested that barbadin, a specific inhibitor of the βarrestin-βadaptin interaction, blocked the Amph-induced increase of hD2R. Taken together our data suggest that prolonged exposures to Amph decrease or increase the endogenous hD2R at the cellular membrane in HEK293 cells expressing or lacking hDAT, respectively. Considering that this drug is often consumed for prolonged periods, during which tolerance develops, our data suggest that even in absence of DAT or dopamine, Amph can still alter D2R distribution and function.

scholarly journals Influence of phosphorylation on isoform composition and function of a microtubule-associated protein from developing Artemia

1995 ◽  
Vol 307 (2) ◽  
pp. 419-424 ◽  
Author(s):  
J Zhang ◽  
T H Macrae
Keyword(s):  
Brine Shrimp ◽  
Electrophoretic Analysis ◽  
Immunofluorescent Staining ◽  
Mobility Shift ◽  
Western Blots ◽  
Microtubule Associated Proteins ◽  
Apparent Homogeneity ◽  
Associated Proteins ◽  
And Function

A novel 49 kDa protein, which exhibits nucleotide-dependent cross-linking of microtubules in vitro and localizes to ordered microtubule arrays by immunofluorescent staining, has been purified to apparent homogeneity from the brine shrimp, Artemia. Electrophoretic analysis involving isoelectric focusing and two-dimensional gels, supplemented by staining of Western blots with affinity-purified antibody, revealed that the 49 kDa protein consists of five isoforms with pI values of 6.0-6.2. The amount of 49 kDa protein increased slightly, but its isoform composition did not change significantly, during development of Artemia gastrula to third-instar larvae. Treatment with alkaline phosphatase caused the 49 kDa protein to undergo a mobility shift on gel electrophoresis, and, by use of an antibody to phosphoserine, at least two isoforms of the protein were shown to be phosphorylated. The serine phosphate, presumably added by a post-translational mechanism, did not influence binding of the 49 kDa protein to microtubules. Under conditions in which microtubules were cross-linked, the 49 kDa protein failed to interact with actin filaments. Our results demonstrate that the 49 kDa protein, like other structural microtubule-associated proteins such as tau and MAP2, is composed of several isoforms, some of which are phosphorylated. This protein has the potential to regulate the spatial distribution of microtubules within cells but does not link microfilaments to one another or to microtubules.

Ultrastructural Changes in Tumor Cells During Human Peripheral Blood Monocyte-Mediated Cytotoxicity

1981 ◽  
Vol 39 ◽  
pp. 452-453
Author(s):  
K. E. Muse ◽  
D. G. Fischer ◽  
H. S. Koren
Keyword(s):  
Target Cell ◽  
Human Peripheral Blood ◽  
Mononuclear Phagocytes ◽  
Ultrastructural Changes ◽  
Target Cells ◽  
Limited Information ◽  
Blood Monocytes ◽  
Tumoricidal Activity ◽  
Activated State

Mononuclear phagocytes, a pluripotential cell line, manifest an array of basic extracellular functions. Among these physiological regulatory functions is the expression of spontaneous cytolytic potential against tumor cell targets.The limited observations on human cells, almost exclusively blood monocytes, initially reported limited or a lack of tumoricidal activity in the absence of antibody. More recently, freshly obtained monocytes have been reported to spontaneously impair the biability of tumor target cells in vitro (Harowitz et al., 1979; Montavani et al., 1979; Hammerstrom, 1979). Although the mechanism by which effector cells express cytotoxicity is poorly understood, discrete steps can be distinguished in the process of cell mediated cytotoxicity: recognition and binding of effector to target cells,a lethal-hit stage, and subsequent lysis of the target cell. Other important parameters in monocyte-mediated cytotoxicity include, activated state of the monocyte, effector cell concentrations, and target cell suseptibility. However, limited information is available with regard to the ultrastructural changes accompanying monocyte-mediated cytotoxicity.

Molecular architecture and functions of the neuronal cytoskeleton

1990 ◽  
Vol 48 (3) ◽  
pp. 2-3
Author(s):  
Nobutaka Hirokawa
Keyword(s):  
Major Elements ◽  
Molecular Structures ◽  
Organelle Transport ◽  
Molecular Architecture ◽  
Neuronal Morphogenesis ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
And Function ◽  
Small Clusters

In this symposium I will present our studies about the molecular architecture and function of the cytomatrix of the nerve cells. The nerve cell is a highly polarized cell composed of highly branched dendrites, cell body, and a single long axon along the direction of the impulse propagation. Each part of the neuron takes characteristic shapes for which the cytoskeleton provides the framework. The neuronal cytoskeletons play important roles on neuronal morphogenesis, organelle transport and the synaptic transmission. In the axon neurofilaments (NF) form dense arrays, while microtubules (MT) are arranged as small clusters among the NFs. On the other hand, MTs are distributed uniformly, whereas NFs tend to run solitarily or form small fascicles in the dendrites Quick freeze deep etch electron microscopy revealed various kinds of strands among MTs, NFs and membranous organelles (MO). These structures form major elements of the cytomatrix in the neuron. To investigate molecular nature and function of these filaments first we studied molecular structures of microtubule associated proteins (MAP1A, MAP1B, MAP2, MAP2C and tau), and microtubules reconstituted from MAPs and tubulin in vitro. These MAPs were all fibrous molecules with different length and formed arm like projections from the microtubule surface.

Role of the 807 C/T Polymorphism of the α2 Gene in Platelet GP Ia Collagen Receptor Expression and Function

1999 ◽  
Vol 81 (06) ◽  
pp. 951-956 ◽  
Author(s):  
J. Corral ◽  
R. González-Conejero ◽  
J. Rivera ◽  
F. Ortuño ◽  
P. Aparicio ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Cell Types ◽  
Receptor Expression ◽  
Case Control Studies ◽  
Platelet Surface ◽  
Vitro Analysis ◽  
And Function ◽  
In Vitro Analysis

SummaryThe variability of the platelet GP Ia/IIa density has been associated with the 807 C/T polymorphism (Phe 224) of the GP Ia gene in American Caucasian population. We have investigated the genotype and allelic frequencies of this polymorphism in Spanish Caucasians. The T allele was found in 35% of the 284 blood donors analyzed. We confirmed in 159 healthy subjects a significant association between the 807 C/T polymorphism and the platelet GP Ia density. The T allele correlated with high number of GP Ia molecules on platelet surface. In addition, we observed a similar association of this polymorphism with the expression of this protein in other blood cell types. The platelet responsiveness to collagen was determined by “in vitro” analysis of the platelet activation and aggregation response. We found no significant differences in these functional platelet parameters according to the 807 C/T genotype. Finally, results from 3 case/control studies involving 302 consecutive patients (101 with coronary heart disease, 104 with cerebrovascular disease and 97 with deep venous thrombosis) determined that the 807 C/T polymorphism of the GP Ia gene does not represent a risk factor for arterial or venous thrombosis.

Type I Interferons promote maintenance and function of follicular T helper cells in vitro

2019 ◽  
Author(s):  
S Ehrlich ◽  
K Wild ◽  
M Smits ◽  
K Zoldan ◽  
M Hofmann ◽  
...  
Keyword(s):  
T Helper Cells ◽  
Type I Interferons ◽  
Type I ◽  
T Helper ◽  
Helper Cells ◽  
Follicular T Helper Cells ◽  
And Function
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