scholarly journals Characterization of bovine tracheobronchial phenol sulphotransferase cDNA and detection of mRNA regulation by cortisol

1995 ◽  
Vol 311 (1) ◽  
pp. 209-217 ◽  
Author(s):  
S J Schauss ◽  
T Henry ◽  
R Palmatier ◽  
L Halvorson ◽  
R Dannenbring ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cdna Library ◽  
Bronchial Epithelial Cell ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Coding Region ◽  
Culture Model ◽  
Steady State Levels ◽  
Tracheal Epithelial ◽  
High Level

Phenol sulphotransferases esterify both endogenous and foreign hydroxylated aromatic compounds with sulphate. Since these enzymes participate in both hormone and drug metabolism, elucidating their regulation at both the enzymic and molecular levels may provide new understanding in several metabolic pathways. The primary structure of a bovine phenol sulphotransferase has been determined by isolation of the corresponding cDNA. Two partial bovine cDNAs were first isolated by probing a tracheal epithelial cell lambda gt11 cDNA library with a rat phenol sulphotransferase cDNA. These clones provided the sequences of the 5′ and 3′ ends of the predicted coding region. A contiguous cDNA was subsequently isolated by PCR using 5′ and 3′ oligonucleotide primers and the cDNA library as the template. The sequence of the resulting approx. 1 kbp cDNA predicted an amino acid sequence that included sequences determined for several tryptic peptides of the purified protein. Antiserum directed to a synthetic N-terminal peptide predicted by the cDNA sequence showed reactivity with the purified enzyme. High-level Trc-promoter-driven expression of the recombinant bovine enzyme was achieved in Escherichia coli. The bovine cDNA was used to determine relative steady-state levels of phenol sulphotransferase transcripts in bovine lung tissues; distal lung parenchymal RNA levels were 6-10-fold greater than those in tracheobronchial epithelium. Using a bronchial epithelial cell culture model, however, cortisol was observed to increase mRNA levels by 5-fold in both a dose- and time-dependent manner; this corresponds to previously reported glucocorticoid stimulation of phenol sulphotransferase activity in this system [Beckmann, Illig and Bartzatt (1994) J. Cell Physiol. 160, 603-610].

scholarly journals Transcriptional Response of Multidrug-Resistant Klebsiella pneumoniae Clinical Isolates to Ciprofloxacin Stress

2021 ◽  
Vol 2021 ◽  
pp. 1-6
Author(s):  
Roman Farooq Alvi ◽  
Bilal Aslam ◽  
Muhammad Hidayat Rasool ◽  
Saima Muzammil ◽  
Abu Baker Siddique ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Stress Responses ◽  
Genetic Resistance ◽  
Transcriptional Response ◽  
Multidrug Resistant ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Isolation And Identification ◽  
Concentration Dependent Manner ◽  
High Level

Background. The term “persisters” refers to a small bacterial population that persists during treatment with high antibiotic concentration or dose in the absence of genetic resistance. The present study was designed to investigate the transcriptional response in indigenous Klebsiella pneumoniae under the ciprofloxacin stress. Methods. Isolation and identification of K. pneumoniae were carried out through standard microbiological protocols. The characterization of quinolone resistance was performed by estimating the quinolone susceptibility testing, MIC estimation, and detecting the QRDR and PMQR. Transcriptional response of the isolates to ciprofloxacin was determined using qPCR. Results. Among 34 isolates, 23 (67%) were resistant to ciprofloxacin. Both QRDR (gyrA and gyrB) and PMQR (qnrA, qnrB, and qnrS) were detected in the isolates, and all were found resistant to ciprofloxacin. The mRNA levels of both mutS and euTu under the influence of ciprofloxacin were significantly increased. On ciprofloxacin exposure, the mRNA levels of the DNA damage response element (mutS) were raised in a time-dependent fashion. K. pneumoniae showed high-level resistance to ciprofloxacin in the presence of mutations in QRDR and PMQR genes. Conclusion. The transcriptional response revealed the upregulation of DNA repair and protein folding elements (mutS and euTu) in ciprofloxacin stress and delayed cell division. The ciprofloxacin was found to trigger various stress responses in a time- and concentration-dependent manner.

LPS-induced neutrophilic inflammation and Bcl-2 expression in metaplastic mucous cells

2003 ◽  
Vol 285 (2) ◽  
pp. L405-L414 ◽  
Author(s):  
Jennifer E. Foster ◽  
Katherine Gott ◽  
Mark R. Schuyler ◽  
Wieslaw Kozak ◽  
Yohannes Tesfaigzi
Keyword(s):  
Epithelial Cell ◽  
Inflammatory Responses ◽  
Mucous Cell ◽  
Epithelial Cell Line ◽  
Mrna Levels ◽  
Mucous Cells ◽  
Tracheal Epithelial Cell ◽  
Neutrophilic Inflammation ◽  
Tracheal Epithelial ◽  
Cytochrome P 450

Our previous studies show that Bcl-2, a regulator of apoptosis, may be involved in the reduction of mucous cell metaplasia (MCM) during recovery from inflammatory responses. The present study was to determine whether neutrophilic inflammation mediates Bcl-2 expression in mucous cells. Rats were intratracheally instilled with 50–1,000 μg of LPS. The number of neutrophils recovered by bronchoalveolar lavage (BAL) increased with the dose of LPS, and the percentage of Bcl-2-expressing cells increased with the numbers of neutrophils in the BAL. Depletion of neutrophils did not reduce MCM, but the percentage of Bcl-2-positive cells increased 1.8-fold in neutrophil-depleted compared with controls. Injection of rats with bezafibrate, an inducer of cytochrome P-450, doubled the number of neutrophils in the BAL, decreased MCM twofold compared with vehicle-injected controls, and reduced Bcl-2 expression. Bcl-2 mRNA levels decreased in a tracheal epithelial cell line treated with bezafibrate. These data demonstrate that Bcl-2 expression is independent of the number of neutrophils in the BAL and that bezafibrate may directly reduce Bcl-2 expression in epithelial cells.

scholarly journals Allyl Isothiocyanate Increases MRP1 Function and Expression in a Human Bronchial Epithelial Cell Line

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Dian-lei Wang ◽  
Chen-yin Wang ◽  
Yin Cao ◽  
Xian Zhang ◽  
Xiu-hua Tao ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Protein Expression ◽  
Cell Line ◽  
Allyl Isothiocyanate ◽  
Bronchial Epithelial Cell ◽  
Epithelial Cell Line ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Bronchial Epithelial ◽  
Mrp1 Protein

Multidrug resistance-associated protein 1 (MRP1), a member of the ATP-binding cassette (ABC) superfamily of transporters, plays an important role in normal lung physiology by protecting cells against oxidative stress and toxic xenobiotics. The present study investigates the effects of allyl isothiocyanate (AITC) onMRP1mRNA and MRP1 protein expression and transporter activity in the immortalised human bronchial epithelial cell line 16HBE14o-.MRP1mRNA and MRP1 protein expression in 16HBE14o- cells that were treated with allyl isothiocyanate were analysed by real-time PCR assay and Western blotting. The transport of carboxyfluorescein, a known MRP1 substrate, was measured by functional flow cytometry to evaluate MRP1 activity. Treatment with AITC at concentrations of 5–40 μM increased MRP1 protein levels in a concentration-dependent manner. AITC treatments at concentrations of 1–40 μM caused concentration-dependent increases inMRP1mRNA levels that were up to seven times greater than the levels found in control cells. Finally, AITC treatment at concentrations of 5–40 μM significantly increased MRP1-dependent efflux in 16HBE14o- cells. These results suggest that AITC can increase the expression and activity of MRP1 in 16HBE14o- cells in a concentration-dependent manner. The upregulation of MRP1 activity and expression by AITC could produce therapeutic effects in the treatment of lung disease.

Differential inhibition of inflammatory cytokine release from cultured alveolar macrophages from smokers and non smokers by No2

1997 ◽  
Vol 16 (10) ◽  
pp. 577-588 ◽  
Author(s):  
Tiziana Dandrea ◽  
Ba Tu ◽  
Anders Blomberg ◽  
Thomas Sandström ◽  
Magnus Sköld ◽  
...  
Keyword(s):  
Steady State ◽  
Alveolar Macrophages ◽  
Exposure Model ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Tnf Α ◽  
Dependent Inhibition ◽  
Steady State Levels ◽  
Dose Dependent

Human alveolar macrophages (AMs) obtained from smokers and non-smokers by bronchoalveolar lavage (BAL) were subjected to various concentrations of NO2 in an inverted monolayer exposure model. Culture super natants were collected 4 h after the exposure and assayed for secreted TNF-α, IL-1β, IL-8 and MIP-1α. The steady state levels of the mRNAs for these cytokines were also analysed in the cells. The adherence of BAL cells to plastic prior to exposure to the gas elevated the steady state mRNA levels of all four cytokines tested in smoker's cells and that of TNF-α and IL-1β, but not IL-8 (MIP-1α not tested), in non-smoker's cells. Interestingly, adherent cells from non-smokers released circa 15-, 3-,1.5- and 3-fold the amounts of IL-1β, IL-8, TNF-α and MIP-1α, respectively, than smoker's cells during control incubation or exposure to air. A 20 min exposure to NO2 (5 or 20 p.p.m.) did not increase the secretion of any of the cytokines from either cell type. In contrast, NO2 caused a concentration- dependent inhibition of the secretion of all cytokines except IL-1β from smoker's cells. Additionally, NO2 greatly diminished the release of all cytokines in response to further treatment with lipopolysaccharide (LPS). In contrast, only the secretion of TNF-α from non-smoker's cells was inhibited by the gas in a concentration- dependent manner, whilst LPS-induced secretion of the cytokines was not affected by the gas. The steady state levels of the respective mRNAs for each of the cytokines were not significantly affected in smoker's cells by exposure to NO2, except for a negative, dose-dependent trend in the case of TNF-α. Nitrogen dioxide also failed to elevate the levels of the mRNAs in non-smoker's cells but, again, tended to diminish the levels, particularly of IL-1β mRNA. However, exposure to the gas inhibited LPS- induced accumulation of cytokine mRNAs in smoker's cells only. The data suggest that macrophage-derived cytokine mediators of the sepsis response may not play a role in the generation of NO2-induced inflammation in the human lung. Conversely, the gas seems to non-specifically inhibit the release and/or production of cytokines, particularly from smoker's cells, at the post-transcrip tional level, and impairs the ability of the cells to increase the transcription and release of the cytokines in response to bacterial LPS. The fact that NO2 seriously impaired the already diminished capacity of smoker's cells to release several important pro-inflammatory cytokines, both under control conditions and in response to LPS, strongly suggest that the inhalation of NO2 in cigarette smoke may contribute to impairing host defence against infection in the lung.

scholarly journals Bronchial Epithelial Tet2 Maintains Epithelial Integrity during Acute Pseudomonas aeruginosa Pneumonia

2020 ◽  
Vol 89 (1) ◽  
pp. e00603-20
Author(s):  
Wanhai Qin ◽  
Xanthe Brands ◽  
Cornelis van't Veer ◽  
Alex F. de Vos ◽  
Brendon P. Scicluna ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Bronchial Epithelial Cell ◽  
Mrna Levels ◽  
Wild Type ◽  
Bronchial Epithelial ◽  
Epithelial Integrity ◽  
Content Type ◽  
Protein Levels

ABSTRACTRespiratory epithelial cells are important for pulmonary innate immune responses during Pseudomonas aeruginosa infection. Tet methylcytosine dioxygenase 2 (Tet2) has been implicated in the regulation of host defense by myeloid and lymphoid cells, but whether Tet2 also contributes to epithelial responses during pneumonia is unknown. The aim of this study was to investigate the role of bronchial epithelial Tet2 in acute pneumonia caused by P. aeruginosa. To this end, we crossed mice with Tet2 flanked by two Lox-P sites (Tet2fl/fl mice) with mice expressing Cre recombinase under the bronchial epithelial cell-specific Cc10 promoter (Cc10Cre mice) to generate bronchial epithelial cell-specific Tet2-deficient (Tet2fl/fl Cc10Cre) mice. Six hours after infection with P. aeruginosa,Tet2fl/fl Cc10Cre and wild-type mice had similar bacterial loads in bronchoalveolar lavage fluid (BALF). At this time point, Tet2fl/fl Cc10Cre mice displayed reduced mRNA levels of the chemokines Cxcl1, Cxcl2, and Ccl20 in bronchial brushes. However, Cxcl1, Cxcl2, and Ccl20 protein levels and leukocyte recruitment in BALF were not different between groups. Tet2fl/fl Cc10Cre mice had increased protein levels in BALF after infection, indicating a disturbed epithelial barrier function, which was corroborated by reduced mRNA expression of tight junction protein 1 and occludin in bronchial brushes. Differences detected between Tet2fl/fl Cc10Cre and wild-type mice were no longer present at 24 h after infection. These results suggest that bronchial epithelial Tet2 contributes to maintaining epithelial integrity by enhancing intracellular connections between epithelial cells during the early phase of P. aeruginosa pneumonia.

scholarly journals Potential Mechanisms of an Antiadenomyosis Chinese Herbal Formula Shaoyao-Gancao Decoction in Primary Cell Culture Model

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Yong-Ge Guan ◽  
Jin-Bin Liao ◽  
Kun-Yin Li ◽  
Yu-Cui Li ◽  
Yang Song ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Culture Model ◽  
Proliferation And Apoptosis ◽  
Medicine Prescription ◽  
Chinese Medicine Prescription ◽  
Induced Apoptosis ◽  
Dose Dependent

Background. Shaoyao-Gancao Decoction (SGD), a well-known traditional Chinese medicine prescription, has been widely used to treat adenomyosis, dysmenorrhea, abdominal pain, and inflammation in Asia. However, the mechanism underlying the effectiveness of SGD in the treatment of adenomyosis still remains elusive. The present study aimed to investigate the bioactivity of SGD and its underlying molecular mechanisms using cultured human adenomyosis-derived cells.Methods. Human adenomyosis-derived cells were treated with SGD and its major constituents (paeoniflorin and liquiritin)in vitro. Effects of SGD, paeoniflorin, and liquiritin on cell proliferation and apoptosis were examined by MTT assay and flow cytometry analyses. The effects of SGD, paeoniflorin, and liquiritin on the production of PGE2and PGF2αwere assayed using ELISA. ER-αand OTR mRNA expression levels were also evaluated by real-time qRT-PCR.Results. SGD, paeoniflorin, and liquiritin inhibited proliferation and induced apoptosis of human adenomyosis-derived cells in a dose-dependent manner. SGD and paeoniflorin significantly reduced the PGE2and PGF2αproduction. Furthermore, they remarkably decreased the mRNA levels of ER-αand OTR.Conclusions. The results of this study provide possible mechanisms for the bioactivity of SGD for treating adenomyosis and contribute to the ethnopharmacological knowledge about this prescription.

Primary structure of rat HGF receptor and induced expression in glomerular mesangial cells

1996 ◽  
Vol 271 (3) ◽  
pp. F679-F688 ◽  
Author(s):  
Y. Liu ◽  
E. M. Tolbert ◽  
A. M. Sun ◽  
L. D. Dworkin
Keyword(s):  
Mesangial Cells ◽  
Rat Kidney ◽  
Receptor Gene ◽  
Receptor Expression ◽  
Tyrosine Kinase Receptor ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Glomerular Mesangial Cells ◽  
Coding Region ◽  
Simultaneous Induction

The c-met protooncogene encodes a transmembrane tyrosine kinase receptor for hepatocyte growth factor (HGF). It has been widely suggested that HGF and its receptor constitute a paracrine signaling system, in which mesenchymally derived cells produce ligand that binds to the receptor predominantly expressed on cells of epithelial origin. In this study, we have isolated and completely sequenced the entire coding region of c-met cDNA from the rat kidney. The nucleotide sequence of the rat c-met cDNA revealed that the HGF receptor is encoded within single open-reading frame as 190 kDa of a transmembrane glycoprotein consisting of 1,382 amino acids. Determination of c-met mRNA levels in various tissues revealed a widespread expression of c-met with the highest levels in kidney, lung, and liver. We found simultaneous induction of both HGF and its receptor gene expression by interleukin-6 (IL-6) in primary cultured rat glomerular mesangial cells. The expression of HGF and c-met was remarkably stimulated following incubation of rat mesangial cells with IL-6, in a time- and dose-dependent manner Our data suggest that autocrine action of HGF may be achieved in vivo through simultaneous induction of both HGF and its receptor expression in renal mesenchymal cells.

scholarly journals Rule of UA on Cardiac Myocytes Uric Acid Differently Influence the Oxidative Damage Induced by Acute Exposure of High Level of Glucose in Chicken Cardiac Myocytes

2020 ◽  
Vol 7 ◽  
Author(s):  
Xiaolong Sun ◽  
Hongchao Jiao ◽  
Jingpeng Zhao ◽  
Xiaojuan Wang ◽  
Hai Lin
Keyword(s):  
Uric Acid ◽  
Oxidative Damage ◽  
Cell Viability ◽  
Cardiac Myocytes ◽  
High Glucose ◽  
Acute Exposure ◽  
Related Factor ◽  
Mrna Levels ◽  
Dependent Manner ◽  
High Level

Background: Uric acid (UA) is a potent scavenger of oxidants in mammalian and avian species. In humans, hyperglycemia with simultaneous hyperuricemia may exert additional damage to the cardiovascular system. Chickens naturally have hyperglycemia (10.1–11.0 mmol/L) and hyperuricemia (100–900 μmol/L), which makes them an interesting model.Methods: The aim of this study was to investigate the effects of UA on the oxidative damage induced by acute exposure of high level of glucose in chicken cardiac myocytes.Results: Cell viability and the concentrations of thiobarbituric acid reactive substance (TBARS) were decreased by glucose treatment in a dose- and time-dependent manner. After acute exposure to high level of glucose (300 mM), a moderate level of UA (300 μM) increased cell viability and reduced TBARS and glutathione (GSH) content. Compared to the control or to independent high glucose (300 mM) or UA (1,200 μM) treatment, the concurrent treatment of high glucose and high UA significantly increased the TBARS, protein carbonyl contents, and ROS concentration, whereas it decreased the cell viability, superoxide dismutase (SOD) activity, and GSH content. In the presence of high glucose and UA, the nucleic protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2) was decreased and the mRNA levels of the genes cat, sod1, sod2, gss, and gclc were downregulated.Conclusion: In conclusion, acute exposure of high level of glucose induced oxidative damage in the cardiac myocytes of chicken. The present result suggests that an adequate level of uric acid is helpful in alleviating the acute oxidative damage that is induced by high glucose, whereas the inhibition of the Nrf2 pathway by a high level of uric acid may render the cardiac myocytes more vulnerable to suffering from oxidative damage.

Molecular cloning of metallothionein cDNA and analysis of metallothionein gene expression in winter flounder tissues

1989 ◽  
Vol 67 (10) ◽  
pp. 2520-2527 ◽  
Author(s):  
King Ming Chan ◽  
William S. Davidson ◽  
Choy L. Hew ◽  
Garth L. Fletcher
Keyword(s):  
Cdna Library ◽  
Untranslated Region ◽  
Cadmium Chloride ◽  
Gill Filament ◽  
Winter Flounder ◽  
Mrna Levels ◽  
Pseudopleuronectes Americanus ◽  
Coding Region ◽  
Liver And Kidney ◽  
Time Required

Investigations into the precise role played by metallothionein (MT) in heavy-metal metabolism have been hampered by difficulties in positively identifying and quantifying MT in fish tissues. This study describes the development of an antisense MT RNA (cRNA) probe that will enable MT mRNA levels to be measured with a high degree of specificity and precision. Cadmium chloride administration induces the producton of MT mRNA in the liver and kidney of winter flounder (Pseudopleuronectes americanus). Poly(A)+ RNA purified from liver samples of winter flounder after cadmium chloride injections was used to construct a cDNA library. Several recombinant clones made complementary to MT mRNA were selected from this cDNA library by an oligonucleotide derived from the N-terminal amino acid sequence of winter flounder metallothionein. Sequence analysis of two of the cDNA inserts gave the structure of the entire 3′ untranslated region, a coding region corresponding to winter flounder MT and 49 nucleotides of the 5′ untranslated region. One of the flounder MT cDNAs, pWFMTC4, was subcloned into a RNA probe plasmid and transcribed to produce antisense MT RNA (cRNA). The MT cRNA was then used to detect the induction of MT mRNA production in the liver of winter flounder, following the administration of Cu2+, Zn2+, Cd2+, Pb2+, and Hg2+. The time required for the induction of hepatic MT mRNA by a single injection of Cd2+ was approximately 96 h. Dexamethasone did not induce an increase of MT mRNA in any of the winter flounder tissues examined (liver, kidney, heart, brain, intestinal scrape, and gill filament), whereas Cd2+ induced MT mRNA in all of the tissues except brain, where the constitutive level of expression was high.

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