scholarly journals Analysis of Nitroxide-Based Distance Measurements in Cell Extracts and in Cells by Pulsed ESR Spectroscopy

ChemPhysChem ◽  
2017 ◽  
Vol 18 (12) ◽  
pp. 1653-1660 ◽  
Author(s):  
Matthew J. Lawless ◽  
Amit Shimshi ◽  
Timothy F. Cunningham ◽  
Monica N. Kinde ◽  
Pei Tang ◽  
...  
Keyword(s):  
Esr Spectroscopy ◽  
Distance Measurements ◽  
Cell Extracts ◽  
In Cells

Nucleotide-Independent Copper(II)-Based Distance Measurements in DNA by Pulsed ESR Spectroscopy

2017 ◽  
Vol 129 (8) ◽  
pp. 2147-2149 ◽  
Author(s):  
Matthew J. Lawless ◽  
Jessica L. Sarver ◽  
Sunil Saxena
Keyword(s):  
Esr Spectroscopy ◽  
Distance Measurements

beta-Carotene is Accumulated, Metabolized, and Possibly Converted to Retinol in Human Breast Carcinoma Cells (MCF-7)

2004 ◽  
Vol 74 (3) ◽  
pp. 171-177 ◽  
Author(s):  
Torres ◽  
Borojevic ◽  
Trugo
Keyword(s):  
Breast Carcinoma ◽  
Beta Carotene ◽  
Human Breast Carcinoma ◽  
Breast Carcinoma Cell Line ◽  
Human Breast ◽  
Cell Extracts ◽  
Human Breast Carcinoma Cells ◽  
Dose Dependent ◽  
Mcf 7 ◽  
In Cells

The aims of the present study were to investigate the uptake, accumulation, and metabolism of beta-carotene by the human breast carcinoma cell line MCF-7. Beta-carotene uptake was time- and dose-dependent, and independent of cell polarity. Beta-carotene accumulation in cells was linear as a function of its concentration in medium (1.3–4.1 mumol/L). It was accompanied by increasing amounts of retinol, which accumulated in cells following a sigmoid pattern, and by other four putative metabolites. Beta-apocarotenals, epoxides, endoperoxides, retinal, retinoic acid, and retinyl esters were not detected in cell extracts. Beta-carotene and its metabolites did not induce alterations in cell morphology or subcellular localization of epithelial mucins. Beta-carotene and retinol were released from cells that had previously accumulated beta-carotene, and were further incubated in beta-carotene- and retinol-free medium, but intracellular retinol content remained constant whereas b-carotene decreased. In conclusion, beta-carotene added to culture medium in physiological concentrations (1–6 mumol/L) is taken up and metabolized in MCF-7 cells, and is possibly converted to retinol.

A new approach to distance measurements between two spin labels in the >10 nm range

2017 ◽  
Vol 19 (7) ◽  
pp. 5222-5229 ◽  
Author(s):  
A. Blank
Keyword(s):  
Esr Spectroscopy ◽  
Dipolar Interaction ◽  
Spin Labels ◽  
Interaction Frequency ◽  
Distance Measurements ◽  
New Approach ◽  
Nm Range

ESR spectroscopy can be efficiently used to acquire the distance between two spin labels placed on a macromolecule by measuring their mutual dipolar interaction frequency, as long as the distance is not greater than ∼10 nm.

scholarly journals Ubiquitinated proteins promote the association of proteasomes with the deubiquitinating enzyme Usp14 and the ubiquitin ligase Ube3c

2017 ◽  
Vol 114 (17) ◽  
pp. E3404-E3413 ◽  
Author(s):  
Chueh-Ling Kuo ◽  
Alfred Lewis Goldberg
Keyword(s):  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Proteasome Activity ◽  
Deubiquitinating Enzyme ◽  
Core Particle ◽  
Ubiquitinated Proteins ◽  
Protein Ubiquitination ◽  
Cell Extracts ◽  
Regulatory Particle ◽  
In Cells

In mammalian cells, the 26S proteasomes vary in composition. In addition to the standard 28 subunits in the 20S core particle and 19 subunits in each 19S regulatory particle, a small fraction (about 10–20% in our preparations) also contains the deubiquitinating enzyme Usp14/Ubp6, which regulates proteasome activity, and the ubiquitin ligase, Ube3c/Hul5, which enhances proteasomal processivity. When degradation of ubiquitinated proteins in cells was inhibited, levels of Usp14 and Ube3c on proteasomes increased within minutes. Conversely, when protein ubiquitination was prevented, or when purified proteasomes hydrolyzed the associated ubiquitin conjugates, Usp14 and Ube3c dissociated rapidly (unlike other 26S subunits), but the inhibitor ubiquitin aldehyde slowed their dissociation. Recombinant Usp14 associated with purified proteasomes preferentially if they contained ubiquitin conjugates. In cells or extracts, adding Usp14 inhibitors (IU-1 or ubiquitin aldehyde) enhanced Usp14 and Ube3c binding further. Thus, in the substrate- or the inhibitor-bound conformations, Usp14 showed higher affinity for proteasomes and surprisingly enhanced Ube3c binding. Moreover, adding ubiquitinated proteins to cell extracts stimulated proteasome binding of both enzymes. Thus, Usp14 and Ube3c cycle together on and off proteasomes, and the presence of ubiquitinated substrates promotes their association. This mechanism enables proteasome activity to adapt to the supply of substrates.

Long-Range Distance Measurements on Nucleic Acids in Cells by Pulsed EPR Spectroscopy

2011 ◽  
Vol 50 (22) ◽  
pp. 5070-5074 ◽  
Author(s):  
Ivan Krstić ◽  
Robert Hänsel ◽  
Olga Romainczyk ◽  
Joachim W. Engels ◽  
Volker Dötsch ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Long Range ◽  
Epr Spectroscopy ◽  
Distance Measurements ◽  
Pulsed Epr ◽  
In Cells

scholarly journals THE PHOTOSYNTHETIC APPARATUS OF RHODOSPIRILLUM MOLISCHIANUM

1965 ◽  
Vol 26 (2) ◽  
pp. 395-412 ◽  
Author(s):  
Sarah P. Gibbs ◽  
W. R. Sistrom ◽  
Patricia B. Worden
Keyword(s):  
Cell Membrane ◽  
Photosynthetic Apparatus ◽  
Variable Number ◽  
Electron Microscopic ◽  
Microscopic Appearance ◽  
Whole Cells ◽  
Cell Extracts ◽  
Internal Membrane ◽  
Rhodospirillum Molischianum ◽  
In Cells

By varying the light intensity and temperature during growth it is possible to obtain cultures of Rhodospirillum molischianum in which the specific bacteriochlorophyll contents differ by as much as fivefold. We used such cultures to compare the changes in the electron microscopic appearance of the cells with the changes in the amount and bacteriochlorophyll content of chromatophore material isolated from cell extracts. The cells contained a variable number of internal membranes which are invaginations of the cell membrane. The shape, size, number, and arrangement of the infoldings varied as the specific bacteriochlorophyll content of the cells changed. In cells with little bacteriochlorophyll, the invaginations were mostly tubular. In cells with larger amounts of bacteriochlorophyll, the invaginations were disc-shaped and the discs were appressed together in stacks of 2 to 10 discs each. Variations in the number of discs per stack could be accounted for by a simple statistical model. The average area per disc increased with increasing bacteriochlorophyll content. Quantitative estimations of the relative volumes occupied by membranes in cells with four different bacteriochlorophyll contents showed that the amount of internal membrane alone had no direct relationship with the bacteriochlorophyll content of the cells; however, the total amount of membrane (cell membrane plus internal membrane) was directly proportional to the bacteriochlorophyll content. The specific bacteriochlorophyll content of isolated chromatophore material was proportional to the bacteriochlorophyll content of whole cells; the total amount of chromatophore material was independent of the bacteriochlorophyll content of whole cells. Several possible explanations of this paradoxical discrepancy between the electron microscope observations and the analytical results are discussed.

Two cis-DNA elements involved in myeloid-cell-specific expression and gamma interferon (IFN-gamma) activation of the human high-affinity Fc gamma receptor gene: a novel IFN regulatory mechanism

1993 ◽  
Vol 13 (4) ◽  
pp. 2182-2192
Author(s):  
C Perez ◽  
J Wietzerbin ◽  
P D Benech
Keyword(s):  
Myeloid Cell ◽  
Gamma Interferon ◽  
Cell Type ◽  
Specific Expression ◽  
Ifn Gamma ◽  
High Affinity ◽  
Cell Extracts ◽  
Dna Elements ◽  
Cell Type Specific ◽  
In Cells

The human high-affinity receptor for the constant region of immunoglobulin G (human Fc gamma R1) is encoded by two mRNAs induced selectively by gamma interferon (IFN-gamma) and expressed in cells of myeloid lineage. The cis-DNA element (GRR) previously found to confer IFN-gamma responsiveness to this gene acts as an inducible enhancer and is the target of an IFN-gamma-activated factor(s) (GIRE-BP) in cells of different origins. Although the GRR motif is not related to the DNA elements involved in the regulation of other IFN-stimulated genes, GIRE-BP binding depends on the IFN-gamma-dependent activation of the 91-kDa protein known to be one of the factors of a transcriptional complex activated by IFN-alpha. Deletions of the Fc gamma R1 promoter allowed us to identify a 25-bp element, downstream from the GRR motif, conferring cell-type-specific expression. This element, called MATE (myeloid activating transcription element), is the DNA target for constitutive factors forming two complexes, MATE-BP1 and MATE-BP2. In accordance with the functional analysis, MATE-BP binding activities were detected in extracts prepared from myeloid cell lines such as THP-1, HL-60, and U-937 but not in HeLa cell extracts. The MATE motif is present not only in the promoter of other Fc receptor genes but also in several promoters of genes whose expression is restricted to monocytic cells. Our results suggest that human Fc gamma R1 gene expression in myeloid cells is initiated by the interaction of IFN-gamma-activated factors with cell-type-specific factors through their binding to the GRR and MATE motifs.

scholarly journals Inhibition of Interferon Signaling by Rabies Virus Phosphoprotein P: Activation-Dependent Binding of STAT1 and STAT2

2006 ◽  
Vol 80 (6) ◽  
pp. 2675-2683 ◽  
Author(s):  
Krzysztof Brzózka ◽  
Stefan Finke ◽  
Karl-Klaus Conzelmann
Keyword(s):  
Rabies Virus ◽  
Transcriptional Activation ◽  
Type I ◽  
Interferon Signaling ◽  
Cell Extracts ◽  
Independent Functions ◽  
Stat Signaling ◽  
Infected Cells ◽  
Ifn Γ ◽  
In Cells

ABSTRACT Rabies virus (RV) phosphoprotein P is an interferon (IFN) antagonist counteracting transcriptional activation of type I IFN (K. Brzózka, S. Finke, and K. K. Conzelmann, J. Virol 79:7673-7681, 2005). We here show that RV P in addition is responsible for preventing IFN-α/β- and IFN-γ-stimulated JAK-STAT signaling in RV-infected cells by the retention of activated STATs in the cytoplasm. Expression of IFN-stimulated response element- and gamma-activated sequence-controlled genes was severely impaired in cells infected with RV SAD L16 or in cells expressing RV P protein from transfected plasmids. In contrast, a recombinant RV expressing small amounts of P had lost the ability to interfere with JAK-STAT signaling. IFN-mediated tyrosine phosphorylation of STAT1 and STAT2 was not impaired in RV P-expressing cells; rather, a defect in STAT recycling was suggested by distinct accumulation of tyrosine-phosphorylated STATs in cell extracts. In the presence of P, activated STAT1 and STAT2 were unable to accumulate in the nucleus. Notably, STAT1 and STAT2 were coprecipitated with RV P only from extracts of cells previously stimulated with IFN-α or IFN-γ, whereas in nonstimulated cells no association of P with STATs was observed. This conditional, IFN activation-dependent binding of tyrosine-phosphorylated STATs by RV P is unique for a viral IFN antagonist. The 10 C-terminal residues of P are required for counteracting JAK-STAT signaling but not for inhibition of transcriptional activation of IFN-β, thus demonstrating two independent functions of RV P in counteracting the host's IFN response.

scholarly journals Cholesterol sulphate affects production of steroid hormones by reducing steroidogenic acute regulatory protein level in adrenocortical cells

2007 ◽  
Vol 195 (3) ◽  
pp. 451-458 ◽  
Author(s):  
Teruo Sugawara ◽  
Eiji Nomura ◽  
Nobuhiko Hoshi
Keyword(s):  
Gene Expression ◽  
Steroid Hormones ◽  
Protein Level ◽  
Promoter Activity ◽  
Adrenocortical Cells ◽  
Star Protein ◽  
Cell Extracts ◽  
H295r Cells ◽  
Steroidogenic Acute Regulatory ◽  
In Cells

Steroidogenic acute regulatory (StAR) protein plays a crucial role in the intramitochondrial movement of cholesterol, where P450 side chain cleavage enzyme resides. Cholesterol sulphate (CS), which is present ubiquitously in mammalian tissues, is not only a precursor of sulphated adrenal steroids but also an inhibitor of cholesterol biosynthesis. This study was designed to examine the biological roles of CS in steroidogenesis in adrenocortical cells. Human adrenocortical carcinoma H295R cells were cultured with various amounts of CS. To evaluate steroid hormone synthesis, pregnenolone production in cells was assayed. The amount of pregnenolone produced by H295R cells in culture medium, to which over 50 μg/ml CS was added, was significantly (P<0.05) decreased compared with that produced by control cells. Western blot analysis was performed to determine StAR protein level using whole cell extracts from cells. StAR protein level decreased when the concentration of CS in the medium was 50 μg/ml, whereas the level of glyceraldehyde-3-phosphate dehydrogenase did not change. To examine the mechanism by which StAR gene expression is controlled, we performed RT-PCR and measured promoter activity in cells transfected with pGL2 StAR reporter constructs. StAR mRNA level and promoter activity were decreased in cells. The decrease in StAR protein level is a result of the low StAR gene expression level. In conclusion, CS affects the production of steroid hormones by reducing StAR protein level in adrenocortical cells.

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