scholarly journals A 40,000-dalton protein from Dictyostelium discoideum affects assembly properties of actin in a Ca2+-dependent manner.

1982 ◽  
Vol 93 (1) ◽  
pp. 205-210 ◽  
Author(s):  
S S Brown ◽  
K Yamamoto ◽  
J A Spudich
Keyword(s):  
Dictyostelium Discoideum ◽  
Calcium Ion ◽  
Gel Filtration ◽  
Major Effect ◽  
Dependent Manner ◽  
Actin Assembly ◽  
Experimental Conditions ◽  
Native Form ◽  
Proteolytic Fragment ◽  
Peptide Maps

A 40,000-dalton protein that affects the assembly properties of actin in a Ca2+-dependent manner has been purified from Dictyostelium discoideum. Gel filtration chromatography indicates that the native form of this protein is a monomer. A major effect of this protein is to reduce the sedimentability of F-actin in a stoichiometric fashion. Nearly complete loss of sedimentability is observed at ratios of the 40,000-dalton protein to actin of greater than 1:10. At low stoichiometries, this protein can accelerate the rate of actin assembly under certain experimental conditions. These effects of the 40,000-dalton protein on the actin assembly properties described above require calcium ion. The 40,000-dalton protein does not exert its effects by proteolyzing actin. Furthermore, peptide maps demonstrate that this protein is not a proteolytic fragment of actin.

scholarly journals Epigenetic Modulation of TLR4 Expression by Sulforaphane Increases Anti-Inflammatory Capacity in Porcine Monocyte-Derived Dendritic Cells

Biology ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 490
Author(s):  
Xueqi Qu ◽  
Christiane Neuhoff ◽  
Mehmet Ulas Cinar ◽  
Maren Pröll ◽  
Ernst Tholen ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dendritic Cells ◽  
Epigenetic Modifications ◽  
Dna Demethylation ◽  
Dependent Manner ◽  
Experimental Conditions ◽  
Hdac Activity ◽  
Protein Levels ◽  
Anti Inflammatory ◽  
Epigenetic Modulation

Inflammation is regulated by epigenetic modifications, including DNA methylation and histone acetylation. Sulforaphane (SFN), a histone deacetylase (HDAC) inhibitor, is also a potent immunomodulatory agent, but its anti-inflammatory functions through epigenetic modifications remain unclear. Therefore, this study aimed to investigate the epigenetic effects of SFN in maintaining the immunomodulatory homeostasis of innate immunity during acute inflammation. For this purpose, SFN-induced epigenetic changes and expression levels of immune-related genes in response to lipopolysaccharide (LPS) stimulation of monocyte-derived dendritic cells (moDCs) were analyzed. These results demonstrated that SFN inhibited HDAC activity and caused histone H3 and H4 acetylation. SFN treatment also induced DNA demethylation in the promoter region of the MHC-SLA1 gene, resulting in the upregulation of Toll-like receptor 4 (TLR4), MHC-SLA1, and inflammatory cytokines’ expression at 6 h of LPS stimulation. Moreover, the protein levels of cytokines in the cell culture supernatants were significantly inhibited by SFN pre-treatment followed by LPS stimulation in a time-dependent manner, suggesting that inhibition of HDAC activity and DNA methylation by SFN may restrict the excessive inflammatory cytokine availability in the extracellular environment. We postulate that SFN may exert a protective and anti-inflammatory function by epigenetically influencing signaling pathways in experimental conditions employing porcine moDCs.

scholarly journals Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.

1988 ◽  
Vol 8 (5) ◽  
pp. 1957-1969 ◽  
Author(s):  
R A Shapiro ◽  
D Herrick ◽  
R E Manrow ◽  
D Blinder ◽  
A Jacobson
Keyword(s):  
Steady State ◽  
Dictyostelium Discoideum ◽  
Mrna Decay ◽  
Decay Rates ◽  
Time Dependent ◽  
Translational Efficiency ◽  
Cdna Clones ◽  
Dependent Manner ◽  
Significant Difference ◽  
Kinetics Of

As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.

scholarly journals Neurofascin as a novel target for autoantibody-mediated axonal injury

2007 ◽  
Vol 204 (10) ◽  
pp. 2363-2372 ◽  
Author(s):  
Emily K. Mathey ◽  
Tobias Derfuss ◽  
Maria K. Storch ◽  
Kieran R. Williams ◽  
Kimberly Hales ◽  
...  
Keyword(s):  
Axonal Injury ◽  
Dependent Manner ◽  
Nodes Of Ranvier ◽  
Native Form ◽  
Neuronal Protein ◽  
Axonal Pathology ◽  
Immune Mediated ◽  
Novel Target

Axonal injury is considered the major cause of disability in patients with multiple sclerosis (MS), but the underlying effector mechanisms are poorly understood. Starting with a proteomics-based approach, we identified neurofascin-specific autoantibodies in patients with MS. These autoantibodies recognize the native form of the extracellular domains of both neurofascin 186 (NF186), a neuronal protein concentrated in myelinated fibers at nodes of Ranvier, and NF155, the oligodendrocyte-specific isoform of neurofascin. Our in vitro studies with hippocampal slice cultures indicate that neurofascin antibodies inhibit axonal conduction in a complement-dependent manner. To evaluate whether circulating antineurofascin antibodies mediate a pathogenic effect in vivo, we cotransferred these antibodies with myelin oligodendrocyte glycoprotein–specific encephalitogenic T cells to mimic the inflammatory pathology of MS and breach the blood–brain barrier. In this animal model, antibodies to neurofascin selectively targeted nodes of Ranvier, resulting in deposition of complement, axonal injury, and disease exacerbation. Collectively, these results identify a novel mechanism of immune-mediated axonal injury that can contribute to axonal pathology in MS.

The calcium antagonist diltiazem inhibits calcification enhanced by calcitonin in growth cartilage of rats in ethane-1-hydroxy-1,1-diphosphonate (EHDP)-induced rickets

1986 ◽  
Vol 113 (1) ◽  
pp. 73-81 ◽  
Author(s):  
Masao Eguchi ◽  
Kenichiro Shibata ◽  
Fumio Wada ◽  
Hideya Kawamura ◽  
Takashi Shimauchi ◽  
...  
Keyword(s):  
Calcium Antagonist ◽  
Calcium Ion ◽  
Concomitant Administration ◽  
Proximal Region ◽  
Dependent Manner ◽  
Short Term ◽  
X Ray ◽  
Growth Cartilage ◽  
Term Administration ◽  
Dose Dependent

Abstract. In an animal model of human rickets developed by giving a short-term administration of large doses of EHDP to young rats, concomitant administration of [Asu1,7]eel calcitonin (CT) with EHDP resulted in the promotion of calcification in growth cartilage. In an attempt to clarify the mechanisms related to the accelerated calcification due to CT, the effects of diltiazem, a calcium antagonist, were studied. Diltiazem suppressed, in a dose-dependent manner, the accelerated calcification due to CT in the growth cartilage, as determined by findings on the soft X-ray photos, contact microradiograph and light microscopic histology of the proximal region of the tibia. This suppression was only evident when diltiazem and CT were given concomitantly. If it is assumed that diltiazem inhibits the entry of calcium ion into the cells of growth cartilage, in the same manner as seen in case of smooth muscle and myocardial cells, then our results indicate that intracellular concentrations of calcium might play an important role in the occurrence of accelerated calcification due to CT.

Functional analysis of a stable transcription arrest site in the first intron of the murine adenosine deaminase gene

1993 ◽  
Vol 13 (5) ◽  
pp. 2718-2729
Author(s):  
S F Kash ◽  
J W Innis ◽  
A U Jackson ◽  
R E Kellems
Keyword(s):  
Rna Polymerase Ii ◽  
Adenosine Deaminase ◽  
Functional Characterization ◽  
Gel Filtration ◽  
Point Mutations ◽  
Elongation Factor ◽  
Dependent Manner ◽  
First Intron ◽  
Intron 1

Transcription arrest plays a role in regulating the expression of a number of genes, including the murine adenosine deaminase (ADA) gene. We have previously identified two prominent arrest sites at the 5' end of the ADA gene: one in the first exon and one in the first intron (J. W. Innis and R. E. Kellems, Mol. Cell. Biol. 11:5398-5409, 1991). Here we report the functional characterization of the intron 1 arrest site, located 137 to 145 nucleotides downstream of the cap site. We have determined, using gel filtration, that the intron 1 arrest site is a stable RNA polymerase II pause site and that the transcription elongation factor SII promotes read-through at this site. Additionally, the sequence determinants for the pause are located within a 37-bp fragment encompassing this site (+123 to +158) and can direct transcription arrest in an orientation-dependent manner in the context of the ADA and adenovirus major late promoters. Specific point mutations in this region increase or decrease the relative pausing efficiency. We also show that the sequence determinants for transcription arrest can function when placed an additional 104 bp downstream of their natural position.

scholarly journals All-Trans Retinoic Acid Enhances Matrix Metalloproteinase 2 Expression and Secretion in Human Myeloid Leukemia THP-1 Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Hien Thi Vu ◽  
Thi Xoan Hoang ◽  
Jae Young Kim
Keyword(s):  
Retinoic Acid ◽  
Matrix Metalloproteinase ◽  
Calcium Ion ◽  
Myeloid Leukemia ◽  
Leukemia Cell Line ◽  
Matrix Metalloproteinase 2 ◽  
Migration And Invasion ◽  
Dependent Manner ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

All-trans retinoic acid (ATRA) is an effective drug for the induction therapy of acute promyelocytic leukemia. However, the treatment is associated with adverse events such as retinoic acid syndrome (RAS) in some patients, whose histologic characteristics included organ infiltration by leukemic cells. Matrix metalloproteinase 2 (MMP-2) is often upregulated in tumor cells and plays a role in tumor cell migration and invasion by degrading the extracellular matrix. In this study, we examined the possible modulatory effects of ATRA on MMP-2 expression and secretion in human myeloid leukemia cell line THP-1. The cells were treated with various concentrations of ATRA, and MMP-2 expression and secretion were examined. MMP-2 expression and secretion started to increase with ATRA concentration as low as 0.1 nM and gradually increased thereafter. Agonists of retinoic acid receptor (RAR) or retinoid X receptor (RXR) alone could enhance MMP-2 secretion, and RAR or RXR antagonists alone could reverse ATRA-induced MMP-2 secretion. ATRA increased intracellular calcium ion levels, and a calcium-channel blocker inhibited ATRA-induced MMP-2 secretion. Dexamethasone suppressed ATRA-induced MMP-2 secretion. Our results suggest that ATRA enhances MMP-2 expression and secretion in human myeloid leukemia THP-1 cells in a calcium ion dependent manner through RAR/RXR signaling pathways, and this enhanced expression and secretion may be associated with the possible mechanisms of RAS.

scholarly journals Calcium Input Potentiates the Transforming Growth Factor (TGF)-β1-dependent Signaling to Promote the Export of Inorganic Pyrophosphate by Articular Chondrocyte

2011 ◽  
Vol 286 (22) ◽  
pp. 19215-19228 ◽  
Author(s):  
Frederic Cailotto ◽  
Pascal Reboul ◽  
Sylvie Sebillaud ◽  
Patrick Netter ◽  
Jean-Yves Jouzeau ◽  
...  
Keyword(s):  
Growth Factor ◽  
Promoter Activity ◽  
Transforming Growth Factor ◽  
Articular Chondrocytes ◽  
Specific Protein ◽  
Dependent Manner ◽  
Articular Chondrocyte ◽  
Experimental Conditions ◽  
Inorganic Pyrophosphate ◽  
Tgf Β1

Transforming growth factor (TGF)-β1 stimulates extracellular PPi (ePPi) generation and promotes chondrocalcinosis, which also occurs secondary to hyperparathyroidism-induced hypercalcemia. We previously demonstrated that ANK was up-regulated by TGF-β1 activation of ERK1/2 and Ca2+-dependent protein kinase C (PKCα). Thus, we investigated mechanisms by which calcium could affect ePPi metabolism, especially its main regulating proteins ANK and PC-1 (plasma cell membrane glycoprotein-1). We stimulated articular chondrocytes with TGF-β1 under extracellular (eCa2+) or cytosolic Ca2+ (cCa2+) modulations. We studied ANK, PC-1 expression (quantitative RT-PCR, Western blotting), ePPi levels (radiometric assay), and cCa2+ input (fluorescent probe). Voltage-operated Ca2+-channels (VOC) and signaling pathways involved were investigated with selective inhibitors. Finally, Ank promoter activity was evaluated (gene reporter). TGF-β1 elevated cCa2+ and ePPi levels (by up-regulating Ank and PC-1 mRNA/proteins) in an eCa2+ dose-dependent manner. TGF-β1 effects were suppressed by cCa2+ chelation or L- and T-VOC blockade while being mostly reproduced by ionomycin. In the same experimental conditions, the activation of Ras, the phosphorylation of ERK1/2 and PKCα, and the stimulation of Ank promoter activity were affected similarly. Activation of SP1 (specific protein 1) and ELK-1 (Ets-like protein-1) transcription factors supported the regulatory role of Ca2+. SP1 or ELK-1 overexpression or blockade experiments demonstrated a major contribution of ELK-1, which acted synergistically with SP1 to activate Ank promoter in response to TGF-β1. TGF-β1 promotes input of eCa2+ through opening of L- and T-VOCs, to potentiate ERK1/2 and PKCα signaling cascades, resulting in an enhanced activation of Ank promoter and ePPi production in chondrocyte.

scholarly journals Glyoxalase I is a novel nitric-oxide-responsive protein

1999 ◽  
Vol 344 (3) ◽  
pp. 837-844 ◽  
Author(s):  
Atsushi MITSUMOTO ◽  
Kwi-Ryeon KIM ◽  
Genichiro OSHIMA ◽  
Manabu KUNIMOTO ◽  
Katsuya OKAWA ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Culture Medium ◽  
Molecular Mechanisms ◽  
Peptide Mapping ◽  
Glyoxalase I ◽  
Dependent Manner ◽  
Human Endothelial Cells ◽  
Native Form ◽  
No Donors ◽  
Dose Dependent

To clarify the molecular mechanisms of nitric oxide (NO) signalling, we examined the NO-responsive proteins in cultured human endothelial cells by two-dimensional (2D) PAGE. Levels of two proteins [NO-responsive proteins (NORPs)] with different pI values responded to NO donors. One NORP (pI 5.2) appeared in response to NO, whereas another (pI 5.0) disappeared. These proteins were identified as a native form and a modified form of human glyoxalase I (Glox I; EC 4.4.1.5) by peptide mapping, microsequencing and correlation between the activity and the isoelectric shift. Glox I lost activity in response to NO, and all NO donors tested inhibited its activity in a dose-dependent manner. Activity and normal electrophoretic mobility were restored by dithiothreitol and by the removal of sources of NO from the culture medium. Glox I was selectively inactivated by NO; compounds that induce oxidative stress (H2O2, paraquat and arsenite) failed to inhibit this enzyme. Our results suggest that NO oxidatively modifies Glox I and reversibly inhibits the enzyme's activity. The inactivation of Glox I by NO was more effective than that of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), another NO-sensitive enzyme. Thus Glox I seems to be a novel NO-responsive protein that is more sensitive to NO than G3PDH.

scholarly journals Microinjection of Ca++-calmodulin causes a localized depolymerization of microtubules.

1983 ◽  
Vol 97 (6) ◽  
pp. 1918-1924 ◽  
Author(s):  
C Keith ◽  
M DiPaola ◽  
F R Maxfield ◽  
M L Shelanski
Keyword(s):  
Mitotic Spindle ◽  
Calcium Ion ◽  
Molar Ratio ◽  
Free Calcium ◽  
Local Concentration ◽  
Dependent Manner ◽  
Calcium Dependent ◽  
Molar Ratios ◽  
Microtubule Polymerization ◽  
Full Complement

The microinjection of calcium-saturated calmodulin into living fibroblasts causes the rapid disruption of microtubules and stress fibers in a sharply delimited region concentric with the injection site. This effect is specific to the calcium-bearing form of calmodulin; neither calcium-free calmodulin nor calcium ion at similar levels affects the cytoskeleton. If cells have previously been microinjected with calcium-free calmodulin, elevation of their intracellular calcium levels to 25 mM potentiates the disruption of microtubules throughout the cytoplasm. Approximately 400 mM free calcium is required to cause an equivalent disruption in uninjected cells. The level of calmodulin necessary to disrupt the full complement of cellular microtubules is found to be approximately in 2:1 molar ratio to tubulin dimer. These results indicate that calmodulin can be localized within the cytoplasm in a calcium-dependent manner and that it can act to regulate the calcium lability of microtubules at molar ratios that could be achieved locally within the cell. Our results are consistent with the hypothesis that calmodulin may be controlling microtubule polymerization equilibria in areas of high local concentration such as the mitotic spindle.

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