scholarly journals Transient Transcriptional Activation of theVibrio cholerae El Tor Virulence Regulator ToxT in Response to Culture Conditions

1999 ◽  
Vol 67 (5) ◽  
pp. 2178-2183 ◽  
Author(s):  
Ana I. Medrano ◽  
Victor J. DiRita ◽  
Gabriela Castillo ◽  
Joaquin Sanchez
Keyword(s):  
Transcriptional Activation ◽  
Time Course ◽  
Growth Period ◽  
Culture Conditions ◽  
Primer Extension ◽  
Protein Levels ◽  
Reverse Transcription Pcr ◽  
Transient Activation ◽  
El Tor

ABSTRACT Vibrio cholerae El Tor require special in vitro culture conditions, consisting of an initial static growth period followed by shift to shaking (AKI conditions), for expression of cholera toxin (CT) and toxin coregulated pili (TCP). ToxT, a regulator whose initial transcription depends on the ToxR regulator, positively modulates expression of CT and TCP. To help understand control of CT and TCP in El Tor vibrios, we monitored ctxAB and ToxR-dependenttoxT transcription by time course primer extension assays. AKI conditions stimulated CT synthesis with an absence ofctxAB transcription during static growth followed by induction upon shaking. ToxR-dependent toxT transcription was induced at the end of the static growth period but was transient, stopping shortly after shaking was initiated but, interestingly, also if the static phase was prolonged. Immunoblot assays showed that ToxR protein levels were not coincidentally transient, implying a protein on/off switch mechanism for ToxR. Despite the transient activation by ToxR, transcription of ctxAB was maintained during shaking. This finding suggested continued toxT expression, possibly through relay transcription from another promoter. The 12.6-kb distant upstream tcpA promoter responsible for expression of the TCP operon has been proposed to provide an alternate toxTmessage by readthrough transcription. Activation of thetcpA promoter is supported by increased expression of TcpA protein during the shaking phase of the culture. Readthrough transcription of toxT from tcpA would be compatible with reverse transcription-PCR evidence for atoxT mRNA at times when ToxR-dependent transcription was no longer detectable by primer extension.

scholarly journals Complexing of the CD-3 subunit by a monoclonal antibody activates a microtubule-associated protein 2 (MAP-2) serine kinase in Jurkat cells

1989 ◽  
Vol 262 (2) ◽  
pp. 449-456 ◽  
Author(s):  
C Hanekom ◽  
A Nel ◽  
C Gittinger ◽  
A Rheeder ◽  
G Landreth
Keyword(s):  
T Cells ◽  
Time Course ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Jurkat Cells ◽  
Serine Kinase ◽  
Transient Activation ◽  
Normal Human ◽  
Dose Dependent

Treatment of Jurkat T-cells with anti-CD-3 monoclonal antibodies resulted in the rapid and transient activation of a serine kinase which utilized the microtubule-associated protein, MAP-2, as a substrate in vitro. The kinase was also activated on treatment of Jurkat cells with phytohaemagglutinin, but with a different time course. The activation of the MAP-2 kinase by anti-CD-3 antibodies was dose-dependent, with maximal activity observed at concentrations of greater than 500 ng/ml. Normal human E-rosette-positive T-cells also exhibited induction of MAP-2 kinase activity during anti-CD-3 treatment. The enzyme was optimally active in the presence of 2 mM-Mn2+; lower levels of activity were observed with Mg2+, even at concentrations up to 20 mM. The kinase was partially purified by passage over DE-52 Sephacel with the activity eluting as a single peak at 0.25 M-NaCl. The molecular mass was estimated to be 45 kDa by gel filtration. The activation of the MAP-2 kinase was probably due to phosphorylation of this enzyme as treatment with alkaline phosphatase diminished its activity. These data demonstrate that the stimulation of T-cells through the CD-3 complex results in the activation of a novel serine kinase which may be critically involved in signal transduction in these cells.

scholarly journals Evaluation of biomarkers for in vitro prediction of drug-induced nephrotoxicity in RPTEC/TERT1 cells

2020 ◽  
Vol 9 (2) ◽  
pp. 91-100 ◽  
Author(s):  
Xuan Qiu ◽  
Yufa Miao ◽  
Xingchao Geng ◽  
Xiaobing Zhou ◽  
Bo Li
Keyword(s):  
Transcriptional Activation ◽  
Messenger Rna ◽  
Aristolochic Acid ◽  
Kidney Injury ◽  
Drug Induced ◽  
Vitro Assay ◽  
Protein Levels ◽  
Aristolochic Acid I

Abstract There have been intensive efforts to identify in vivo biomarkers that can be used to monitor drug-induced kidney damage before significant impairment occurs. Kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, clusterin, β2-microglobulin and cystatin C (CysC) have been validated as clinical or preclinical biomarkers in urinary and plasma predictive of acute and chronic kidney injuries and diseases. A high-throughput in vitro assay predictive of nephrotoxicity could potentially be implemented in early drug discovery stage to reduce attrition at later stages of drug development. To assess the potential of these known in vivo biomarkers for in vitro evaluation of drug-induced nephrotoxicity, we selected four nephrotoxic agents (cisplatin, cyclosporin, aristolochic acid I and gentamicin) and detected their effects on the protein levels of nephrotoxic biomarkers in RPTEC/TERT1 cells. The protein levels of clusterin, CysC, GSTπ and TIMP-1 significantly increased in the conditioned media of RPTEC/TERT1 cells treated with cisplatin, cyclosporin, aristolochic acid I and gentamicin. The messenger RNA levels of clusterin, CysC, GSTπ and TIMP-1 also increased in RPTEC/TERT1 cells treated with cisplatin, cyclosporin, aristolochic acid I and gentamicin, indicating that drug-induced upregulation involves transcriptional activation. Taken together, the results clearly demonstrate that among the known in vivo nephrotoxic biomarkers, clusterin, CysC, GSTπ and TIMP-1 can be effectively used as in vitro biomarkers for drug-induced nephrotoxicity in RPTEC/TERT1 cells.

scholarly journals CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation

2008 ◽  
Vol 181 (6) ◽  
pp. 959-972 ◽  
Author(s):  
Xueni Li ◽  
Mei Huang ◽  
Huiling Zheng ◽  
Yinyin Wang ◽  
Fangli Ren ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Osteoblast Differentiation ◽  
Degradation Mechanism ◽  
Interacting Protein ◽  
E3 Ligases ◽  
Protein Levels ◽  
C Terminus ◽  
Osteoblast Lineage

Runx2, an essential transactivator for osteoblast differentiation, is tightly regulated at both the transcriptional and posttranslational levels. In this paper, we report that CHIP (C terminus of Hsc70-interacting protein)/STUB1 regulates Runx2 protein stability via a ubiquitination-degradation mechanism. CHIP interacts with Runx2 in vitro and in vivo. In the presence of increased Runx2 protein levels, CHIP expression decreases, whereas the expression of other E3 ligases involved in Runx2 degradation, such as Smurf1 or WWP1, remains constant or increases during osteoblast differentiation. Depletion of CHIP results in the stabilization of Runx2, enhances Runx2-mediated transcriptional activation, and promotes osteoblast differentiation in primary calvarial cells. In contrast, CHIP overexpression in preosteoblasts causes Runx2 degradation, inhibits osteoblast differentiation, and instead enhances adipogenesis. Our data suggest that negative regulation of the Runx2 protein by CHIP is critical in the commitment of precursor cells to differentiate into the osteoblast lineage.

The Regulation of Natural Food Dyes Curcumin on the Amyloidogenic Pathway of APP

2013 ◽  
Vol 781-784 ◽  
pp. 1160-1163
Author(s):  
Xiong Zhang ◽  
Jie Yun Sun ◽  
Hong Mei Zhang ◽  
Lu Si ◽  
Yu Li
Keyword(s):  
Time Course ◽  
Mrna Level ◽  
Time Dependent ◽  
Hek293 Cells ◽  
Natural Food ◽  
Inhibition Effect ◽  
Dose Time ◽  
Protein Levels ◽  
Food Dyes

As a promising prevention and therapeutic intervention in Alzheimer’s disease, natural food dyes curcumin could obviously inhibit the generation of Aβ, but the mechanism is not fully defined. This study aims to investigate the effects of curcumin on the amyloidogenic pathway of APP in vitro. Plasmids APPswe and BACE1-mychis were transiently co-transfected into SH-SY5Y and HEK293 cells. Then, they were treated with curcumin at 0, 1.25, 5, 20 μmol/L for 24 h, or with curcumin at 5μmol/L for 0, 12, 24 and 48 h for the time course assay. Our findings showed that curcumin could inhibit the expression of the APP at mRNA level; the expression of the BACE1 and C99 at mRNA and protein levels, furthermore it could inhibit the generation of Aβ40/42, and those changes were dose-time dependent (p<0.05). Our study indicates that Aβ40/42 generation inhibition effect of curcumin might due to its influence on amyloidogenic pathway. This may provide important experimental basis for AD treatment with curcumin.

scholarly journals A Vibrio cholerae LysR Homolog, AphB, Cooperates with AphA at the tcpPH Promoter To Activate Expression of the ToxR Virulence Cascade

1999 ◽  
Vol 181 (14) ◽  
pp. 4250-4256 ◽  
Author(s):  
Gabriela Kovacikova ◽  
Karen Skorupski
Keyword(s):  
Vibrio Cholerae ◽  
Virulence Genes ◽  
Virulence Gene ◽  
Growth Conditions ◽  
Culture Conditions ◽  
Virulence Gene Expression ◽  
Environmental Stimuli ◽  
High Level ◽  
El Tor

ABSTRACT We describe here a new member of the LysR family of transcriptional regulators, AphB, which is required for activation of the Vibrio cholerae ToxR virulence cascade. AphB activates the transcription of the tcpPH operon in response to environmental stimuli, and this process requires cooperation with a second protein, AphA. The expression of neither aphA or aphB is strongly regulated by environmental stimuli, raising the possibility that the activities of the proteins themselves may be influenced under various conditions. Strains of the El Tor biotype of V. choleraetypically exhibit lower expression of ToxR-regulated virulence genes in vitro than classical strains and require specialized culture conditions (AKI medium) to induce high-level expression. We show here that expression of aphB from the tac promoter in El Tor biotype strains dramatically increases virulence gene expression to levels similar to those observed in classical strains under all growth conditions examined. These results suggest that AphB plays a role in the differential regulation of virulence genes between the two disease-causing biotypes.

scholarly journals The Late Osteoblast/Preosteocyte Cell Line MLO-A5 Displays Mesenchymal Lineage PlasticityIn VitroandIn Vivo

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Dongqing Yang ◽  
Stan Gronthos ◽  
Sandra Isenmann ◽  
Howard A. Morris ◽  
Gerald J. Atkins
Keyword(s):  
Cell Line ◽  
Alcian Blue ◽  
Culture Conditions ◽  
Specific Gene ◽  
Stem Cell Markers ◽  
Osteogenic Cell ◽  
Reverse Transcription Pcr ◽  
Safranin O

The process of osteoblast switching to alternative mesenchymal phenotypes is incompletely understood. In this study, we tested the ability of the osteoblast/preosteocyte osteogenic cell line, MLO-A5, to also differentiate into either adipocytes or chondrocytes. MLO-A5 cells expressed a subset of skeletal stem cell markers, including Sca-1, CD44, CD73, CD146, and CD166. Confluent cultures of cells underwent differentiation within 3 days upon the addition of osteogenic medium. The same cultures were capable of undergoing adipogenic and chondrogenic differentiation under lineage-appropriate culture conditions, evidenced by lineage-specific gene expression analysis by real-time reverse-transcription-PCR, and by Oil Red O and alcian blue (pH 2.5) staining, respectively. Subcutaneous implantation of MLO-A5 cells in a gel foam into NOD SCID mice resulted in a woven bone-like structure containing embedded osteocytes and regions of cartilage-like tissue, which stained positive with both alcian blue (pH 2.5) and safranin O. Together, our findings show that MLO-A5 cells, despite being a strongly osteogenic cell line, exhibit characteristics of skeletal stem cells and display mesenchymal lineage plasticityin vitroandin vivo. These unique characteristics suggest that this cell line is a useful model with which to study aging and disease-related changes to the mesenchymal lineage composition of bone.

Cytochrome c transcriptional activation and mRNA stability during contractile activity in skeletal muscle

1999 ◽  
Vol 277 (1) ◽  
pp. E26-E32 ◽  
Author(s):  
Damien Freyssenet ◽  
Michael K. Connor ◽  
Mark Takahashi ◽  
David A. Hood
Keyword(s):  
Cytochrome C ◽  
Transcriptional Activation ◽  
Mrna Stability ◽  
Contractile Activity ◽  
Chronic Stimulation ◽  
C Protein ◽  
Protein Levels ◽  
Dna Injection ◽  
Cytosolic Factor

We evaluated contractile activity-induced alterations in cytochrome c transcriptional activation and mRNA stability with unilateral chronic stimulation (10 Hz, 3 h/day) of the rat tibialis anterior (TA) muscle for 1, 2, 3, 4, 5, and 7 days ( n = 3–11/group). Transcriptional activation was assessed by direct plasmid DNA injection into the TA with a chloramphenicol acetyltransferase (CAT) reporter gene linked to 326 bp of the cytochrome c promoter. Cytochrome c mRNA in stimulated muscles increased by 1.3- to 1.7-fold above control between 1 and 7 days. Cytochrome c protein was increased after 5 days of stimulation to reach levels that were 1.9-fold higher than control by 7 days. Cytochrome c mRNA stability, determined with an in vitro decay assay, was greater in stimulated TA than in control between 2 and 4 days, likely mediated by the induction of a cytosolic factor. In contrast, cytochrome c transcriptional activation was elevated only after 5 days of stimulation when mRNA stability had returned to control levels. Thus the contractile activity-induced increase in cytochrome c mRNA was due to an early increase in mRNA stability, followed by an elevation in transcriptional activation, leading to an eventual increase in cytochrome c protein levels.

scholarly journals Transcriptional regulation of heme oxygenases by HIF-1α in renal medullary interstitial cells

2001 ◽  
Vol 281 (5) ◽  
pp. F900-F908 ◽  
Author(s):  
Zhi-Zhang Yang ◽  
Ai-Ping Zou
Keyword(s):  
Mrna Expression ◽  
Transcriptional Activation ◽  
Hypoxia Inducible Factor ◽  
Renal Medulla ◽  
Interstitial Cells ◽  
Ferrous Ammonium Sulfate ◽  
Protein Levels ◽  
Ubiquitin Proteasome ◽  
Ferrous Ammonium

The present study was designed to test the hypothesis that hypoxia-inducible factor-1α (HIF-1α)-mediated transcriptional activation contributes to increased expression of heme oxygenase (HO) genes in renal medullary interstitial cells (RMICs). By Northern blot analysis, HO-1 mRNA expression was found to significantly increase in response to reduction of Po2in culture medium. However, HO-2 mRNA was not altered by hypoxia. This hypoxia-induced upregulation of HO-1 mRNA was significantly blocked by HIF-1α inhibition with ferrous ammonium sulfate. To further determine the role of HIF-1α in the activation of HO-1, the inducers of HIF-1α were used to address whether induction of HIF-1α stimulates HO-1 mRNA expression. Both desferrioxamine and CoCl2markedly increased HIF-1α mRNA and protein levels and resulted in the upregulation of HO-1 mRNA but not HO-2. Furthermore, inhibition of HIF-1α degradation by CBZ-LLL, an inhibitor of ubiquitin-proteasome, significantly increased HIF-1α protein and HO-1 mRNA but not HO-2 in these cells. Using cis-element oligodeoxynucleotide transfection to specifically decoy HIF-1α and block HIF-1α binding, increased mRNA expression of HO-1 in response to hypoxia and CoCl2was attenuated. In vitro nuclear run-on assays further confirmed that hypoxia and alterations of HIF-1α mRNA or protein levels significantly affected the formation of HO-1 mRNA. Taken together, our results indicate that HO-1, but not HO-2, is transcriptionally activated by hypoxia through HIF-1α-mediated mechanism in RMICs. This hypoxia-induced transcriptional activation may be one of the important mechanisms mediating increased expression of HO-1 in the renal medulla.

scholarly journals Changes in nucleic acid and protein levels during in vitro germ¬nation and elongation of Lilium longifforum cv. "Ace" polleni

2014 ◽  
Vol 50 (1-2) ◽  
pp. 39-50
Author(s):  
William V. Dashek
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Total Protein ◽  
Time Course ◽  
Lilium Longiflorum ◽  
Protein Levels ◽  
Dna And Rna ◽  
Rna And Dna ◽  
Rna Levels

While changes in nucleic acid and protein levels during germination and subsequent tube elongation have been determined for a number of pollens, they have not been extensively examined for <em>in vitro</em> grown <em>Lilium longiflorum</em>, cv. `Ace' pollen. Nucleic acids and proteins were extracted with cold trichloroacetic acrid (TCA), cold-hot TCA or cold TCA and potassium hydroxide-perchloric acid (KOH-HClO<sub>4</sub>). Following extraction, RNA, DNA and total protein were assayed colorimetrically with orcinol, diphenylamine and Folin-Phenol reagents, respectively. Extraction of 500 x g supernatants with KOH-HClO<sub>4</sub>, yielded less RNA than either of the TCA-extraction procedures which gave similar nucleic acids and protein recoveries. Whereas total protein levels decreased initially and then increased during 36 h, RNA and DNA levels rose throughout the time-course. Precipitation and quaritiation of nucleic acids and protein from homogenized and soaicated 500 x g pellets resulted in time-dependent alterations in levels of macromolecules which differed from those for 500 x g supernatants. Whereas DNA and RNA levels increased and then decreased over 36 h, total protein levels remained constant for 12 h and then declined during the : next 24 h. Addition of the data obtained for 500 x g supernatants to those for 500 x g pellets revealed that total protein levels increased 2.4 times for the first 12 h and thereafter remained constant, that RNA levels increased 9.8 times for the first 12 h and then levelled off and that the DNA content rose more than 5 times over 36 h.

scholarly journals Changes in Functional Glucocorticoid Sensitivity of Isolated Splenocytes Induced by Chronic Psychosocial Stress – A Time Course Study

2021 ◽  
Vol 12 ◽  
Author(s):  
Elena Kempter ◽  
Mattia Amoroso ◽  
Hannah L. Duffner ◽  
Andrea M. Werner ◽  
Dominik Langgartner ◽  
...  
Keyword(s):  
Psychosocial Stress ◽  
Time Course ◽  
Immune Cell ◽  
Underlying Mechanism ◽  
Low Grade ◽  
Cd11b Expression ◽  
Protein Levels ◽  
Chronic Psychosocial Stress ◽  
Splenic Cd11b

Chronic psychosocial stress is a risk factor for the development of numerous disorders, of which most are associated with chronic low-grade inflammation. Given the immunosuppressive effects of glucocorticoids (GC), one underlying mechanism might be the development of stress-induced GC resistance in certain immune cell subpopulations. In line with this hypothesis, male mice exposed to the chronic subordinate colony housing (CSC, 19 days) model develop GC resistance of in vitro lipopolysaccharide (LPS)-stimulated splenocytes, splenomegaly and an increased percentage of splenic CD11b+ cells. Here male C57BL/6N mice were euthanized at different days during CSC, and following 30 days of single housing after stressor termination to assess when CSC-induced splenic GC resistance starts to develop and whether this is a transient effect. Moreover, splenic CD11b, GC receptor (GR) and/or macrophage migration inhibiting factor (MIF) protein levels were quantified at respective days. While mild forms of CSC-induced GC resistance, increased splenic CD11b expression and/or splenomegaly were detectable on days 8 and 9 of CSC, more severe forms took until days 15 and 16 to develop, but normalized almost completely within 30 days following stressor termination (day 51). In contrast, splenic GR expression was decreased in CSC versus single-housed control (SHC) mice at all days assessed. While MIF expression was increased on days 15 and 16 of CSC, it was decreased in CSC versus SHC mice on day 20 despite persisting splenomegaly, increased CD11b expression and functional GC resistance. In summary, our data indicate that GC resistance and CD11b+ cell-mediated splenomegaly develop gradually and in parallel over time during CSC exposure and are transient in nature. Moreover, while we can exclude that CSC-induced reduction in splenic GR expression is sufficient to induce functional GC resistance, the role of MIF in CD11b+ cell-mediated splenomegaly and GC resistance requires further investigation.

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