scholarly journals The mRNA and protein expression of ruminal MCT1 is increased by feeding a mixed hay/concentrate diet compared with hay <i>ad libitum</i> diet (Short Communication)

2011 ◽  
Vol 54 (3) ◽  
pp. 280-286 ◽  
Author(s):  
J. Kuzinski ◽  
M. Röntgen
Keyword(s):  
Protein Expression ◽  
Flow Cytometric Analysis ◽  
High Energy ◽  
Functional Adaptation ◽  
Monocarboxylate Transporter ◽  
Monocarboxylate Transporter 1 ◽  
Rumen Epithelium ◽  
Flow Cytometric ◽  
Mrna And Protein Expression ◽  
Ad Libitum

Abstract. In this study the protein and mRNA expression of Monocarboxylate transporter 1 (MCT1) was evaluated in rumen epithelial cells (REC) obtained from sheep fed hay ad libitum (control, h diet, n=4) or a mixed hay/concentrate diet (h/c diet, n=4) for two weeks. REC were isolated via fractionated trypsination and three groups consisting of fractions 3 to 5=G1, fractions 6 to 8=G2, and fractions 9 and 10=G3 were formed. Using an anti-basal cytokeratin antibody and flow cytometric analysis, the proportion of REC originating from the stratum basale (SB) was determined for each group. In addition, MCT1 mRNA and protein expression was determined by qRT-PCR and Western blot, respectively. Feeding the h/c diet led to a 299±93 % elevation of the number of SB cells known to express the MCT1 protein. This is accompanied by an increased MCT1 mRNA (1.8 to 2.2-fold) and protein (1.3-fold) expression. Thus, an increased number of MCT1 expressing cells and upregulation of ruminal MCT1 protein seem to be components of rumen epithelium functional adaptation to high energy diet.

scholarly journals Evodiamine Inhibits Gastric Cancer Cell Proliferation via PTEN-Mediated EGF/PI3K Signaling Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-15
Author(s):  
Ruichuang Yang ◽  
Jianxia Wen ◽  
Tao Yang ◽  
Chunmei Dai ◽  
Yanling Zhao
Keyword(s):  
Gastric Cancer ◽  
Cell Migration ◽  
Protein Expression ◽  
Flow Cytometric Analysis ◽  
Migration And Invasion ◽  
Pi3k Signaling ◽  
Dependent Manner ◽  
Flow Cytometric ◽  
Mrna And Protein Expression ◽  
Pi3k Signaling Pathway

Aims. In this study, the pharmacological effects and potential molecular mechanisms of evodiamine in treating gastric cancer (GC) were investigated. Methods. GC cells lines of AGS and BGC-823 were treated with evodiamine at various concentrations for different times (24, 48, and 72 h). Inhibition of the proliferation of AGS and BGC-823 cells was assessed using a CCK-8 assay. The morphology of gastric cancer cells was detected by high-content screening (HCS). The apoptosis-inducing effect of evodiamine on AGS and BGC-823 cells was detected by flow cytometric analysis. Cell migration and invasion were detected by Transwell assay. The relative mRNA and protein expression levels of PTEN-mediated EGF/PI3K signaling pathways were investigated via RT-qPCR or western blotting, respectively. Results. Evodiamine substantially inhibited AGS and BGC-823 cells proliferation in a dose- and time-dependent manner. Flow cytometric analysis revealed that evodiamine could induce apoptosis of AGS and BGC-823 cells in a dose-dependent manner. In addition, evodiamine inhibited AGS and BGC-823 cell migration and invasion. Mechanistically, the results demonstrated that evodiamine promoted the relative mRNA and protein expression of PTEN and decreased expression of EGF, EGFR, PI3K, AKT, p-AKT, and mTOR. Most importantly, evodiamine could effectively increase the mRNA and protein expression of PTEN and decrease the protein expression of EGF/PI3K pathway, indicating that evodiamine downregulated EGF/PI3K through the activation of PTEN pathway. Conclusion. Evodiamine inhibited the directional migration and invasion of GC cells by inhibiting PTEN-mediated EGF/PI3K signaling pathway. These findings revealed that evodiamine might serve as a potential candidate for the treatment or prevention of GC.

scholarly journals Modulation of vH+-ATPase is part of the functional adaptation of sheep rumen epithelium to high-energy diet

2012 ◽  
Vol 303 (9) ◽  
pp. R909-R920 ◽  
Author(s):  
Judith Kuzinski ◽  
Rudolf Zitnan ◽  
Elke Albrecht ◽  
Torsten Viergutz ◽  
Monika Schweigel-Röntgen
Keyword(s):  
Young Male ◽  
Fold Increase ◽  
High Energy ◽  
Functional Adaptation ◽  
Subunit Mrna ◽  
B Subunit ◽  
Rumen Epithelium ◽  
Ad Libitum ◽  
Sheep Rumen

Ruminal vacuolar H+-ATPase (vH+-ATPase) activity is regulated by metabolic signals. Thus, we tested whether its localization, expression, and activity were changed by different feeding. Young male sheep ( n = 12) were either fed hay ad libitum (h) or hay ad libitum plus additional concentrate (h/c) for 2 wk. The vH+-ATPase B subunit signal was predominantly found in the cell membrane and cytosol of rumen epithelial cells (REC) with basal/parabasal phenotype. The elevated number (threefold) of these cells in rumen mucosa of h/c-fed sheep reflects a high proliferative capacity and, explains the 2.3-fold increase of the total number of vH+-ATPase-expressing REC. However, in accordance with a 58% reduction of the vH+-ATPase B subunit mRNA expression in h/c-fed sheep, its protein amount per single REC was decreased. Using the fluorescent probe BCECF and selective inhibitors (foliomycin, amiloride), the contribution of vH+-ATPase and Na+/H+ exchanger to intracellular pH (pHi) regulation was investigated. REC isolated from h/c-fed sheep keep their pHi at a significantly higher level (6.91 ± 0.03 vs. 6.74 ± 0.05 in h-fed sheep). Foliomycin or amiloride decreased pHi by 0.16 ± 0.02 and 0.57 ± 0.04 pH units when applied to REC from h-fed sheep, but the effects were markedly reduced (−88 and −33%) after concentrate feeding. Nevertheless, we found that REC proliferation rate and [cAMP]i were reduced after foliomycin-induced vH+-ATPase inhibition. Our results provide the first evidence for a role of vH+-ATPase in regulation of REC proliferation, most probably by linking metabolically induced pHi changes to signaling pathways regulating this process.

scholarly journals Monocarboxylate transporter-1 (MCT1) protein expression in head and neck cancer affects clinical outcome

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Martin Leu ◽  
J. Kitz ◽  
Y. Pilavakis ◽  
S. Hakroush ◽  
H. A. Wolff ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Protein Expression ◽  
Head And Neck ◽  
Cox Regression ◽  
Locally Advanced ◽  
Monocarboxylate Transporter ◽  
Positive Staining ◽  
Monocarboxylate Transporter 1 ◽  
Prognostic Features ◽  
Hpv Status

AbstractTreatment of locally advanced, unresectable head and neck squamous cell carcinoma (HNSCC) often yields only modest results with radiochemotherapy (RCT) as standard of care. Prognostic features related to outcome upon RCT might be highly valuable to improve treatment. Monocarboxylate transporters-1 and -4 (MCT1/MCT4) were evaluated as potential biomarkers. A cohort of HNSCC patients without signs for distant metastases was assessed eliciting 82 individuals eligible whereof 90% were diagnosed with locally advanced stage IV. Tumor specimens were stained for MCT1 and MCT4 in the cell membrane by immunohistochemistry. Obtained data were evaluated with respect to overall (OS) and progression-free survival (PFS). Protein expression of MCT1 and MCT4 in cell membrane was detected in 16% and 85% of the tumors, respectively. Expression of both transporters was not statistically different according to the human papilloma virus (HPV) status. Positive staining for MCT1 (n = 13, negative in n = 69) strongly worsened PFS with a hazard ratio (HR) of 3.1 (95%-confidence interval 1.6–5.7, p < 0.001). OS was likewise affected with a HR of 3.8 (2.0–7.3, p < 0.001). Multivariable Cox regression confirmed these findings. We propose MCT1 as a promising biomarker in HNSCC treated by primary RCT.

129 microRNA-183~96~182 CLUSTER PROMOTE BOVINE GRANULOSA CELL PROLIFERATION THROUGH COORDINATED REGULATION OF FOXO1

2016 ◽  
Vol 28 (2) ◽  
pp. 194
Author(s):  
S. Gebremedhn ◽  
D. Salilew-Wondim ◽  
M. Hoelker ◽  
F. Rings ◽  
C. Neuhoff ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Protein Expression ◽  
Statistical Significance ◽  
Flow Cytometric Analysis ◽  
Cell Counting ◽  
G1 Arrest ◽  
Mirna Cluster ◽  
Mrna And Protein Expression ◽  
Coordinated Regulation

Among other microRNA clusters, we previously showed that the miR-183~96~182 cluster (miR-183, miR-96, and miR-182) is abundantly expressed in bovine granulosa cells (bGC) of preovulatory dominant follicles obtained at the follicular phase of the bovine oestrous cycle. Moreover, this miRNA cluster are validated to coordinately target the Fork head O1 (FOXO1), a subfamily of transcription factors that regulate genes involved in cell proliferation, apoptosis, cell cycle arrest, and metabolism. However, the functional involvement of miR-183~96~182 cluster in bGC function by regulation of FOXO1 is not yet determined. Here, we aimed to investigate the function of miR-183~96~182 cluster in bGC using in vitro cell culture model. For this, bGC were aspirated from ovarian follicles (Ø 3–5 mm) obtained from local abattoir. Cells were plated in 24-well plate (2.5 × 105 cells well–1) in DMEM/F-12 (Sigma, Germany) supplemented with 10% FBS (GIBCO, Grand Island, NY) and 1% penicillin/streptomycin (GIBCO) and incubated at 37°C in 5% CO2. Transfection of bGC with miRNA mimics, inhibitors, FOXO1-siRNA, and appropriate controls (Exiqon, Vedbæk, Denmark) was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Cell proliferation was determined using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technology, Kumamoto, Japan). Cell cycle distribution was determined with flow cytometric analysis. Total RNA was isolated using miRNeasy mini kit (Qiagen, Hilden, Germany), quantification of target gene was performed using qPCR, and data were analysed using ΔΔCT method. Differences in the mean expression values between treatments were analysed with two-tailed Student’s t-test and statistical significance was defined at P ≤ 0.05. Results showed that a sponge effect was observed upon inhibition in individual miRNA of the cluster, which could be attributed to the partial sequence similarity among cluster members. Both FOXO1 mRNA and protein expression were significantly reduced upon transfection of bGC with miR-183~96~182 cluster mimics, while miR-183~96~182 cluster inhibition increased both FOXO1 mRNA and protein expression. Transfection of bGC with miR-183~96~182 mimics promoted cell proliferation, while inhibition tends to slow down proliferation. Furthermore, the proportion of bGC under G0/G1 arrest markedly declined (P < 0.05), while the S and G2/M phases increased in response to miR-183~96~182 mimicking. Selective knockdown of FOXO1 with FOXO1-siRNA significantly reduced FOXO1 mRNA and protein expression. Interestingly, knockdown of FOXO1 showed similar phenotypic effects such as that of miR-183~96~182 mimics transfection, which resulted in elevated bGC proliferation and reduction in the proportion of cells under G0/G1 arrest. In conclusion, overexpression of miR-183~96~182 cluster promote bGC proliferation and G0/G1 to S and G2/M cell cycle transition through coordinated regulation of genes in the FOXO1 signaling axis.

Is Zap-70 Protein Detection by Flow Cytometry a Reliable Tool in CLL Prognostic Evaluation?.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4771-4771
Author(s):  
Magali Le Garff-Tavernier ◽  
Michel Ticchioni ◽  
Rémi Letestu ◽  
Martine Brissard ◽  
Frédéric Davi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
B Cells ◽  
Protein Expression ◽  
B Cell ◽  
Flow Cytometric Analysis ◽  
Protein Detection ◽  
Flow Cytometric ◽  
Mutational Status ◽  
Healthy Donors

Abstract Background : Expression of ZAP-70 protein has been shown to be correlated with mutational status of immunoglobulin heavy-chain variable region (IgVH) genes, a major prognostic factor in CLL. We investigated whether the detection of ZAP-70 protein by flow cytometric analysis using unconjugated and conjugated monoclonal antibodies (mAbs) could be applied securely in the workup of patients with CLL. Methods: Flow cytometric analysis of ZAP-70 protein was performed using the method described by Crespo et al (N Engl J Med2003;348:1764) with minor modifications. Both fresh and cryopreserved mononuclear cells from CLL patients and healthy donors were fixed and permeabilized using Fix and Perm kit (Caltag Laboratories), incubated with anti-ZAP-70 mAb (clone 2F3.2, Upstate Biotechnology) and then revealed with goat antimouse FITC mAb (Immunotech). Finally cells were incubated with CD3-APC, CD56-APC and CD19-PC5. We also tested 3 mAbs conjugated to various fluorochromes: 2F3.2-FITC (Upstate), 1E7.2-PE (eBioscience), 1E7.2-PE or -Alexa 488 (Caltag). ZAP-70 protein detection in B-cells was expressed either as a percentage of its expression in the T and NK-cells or as a ratio (R) of T-cell mean cell fluorescence (MCF) to B-cell MCF. Western blotting of protein lysates from purified B-cells was carried out to control results obtained by cytometry in 55 samples. Mutational status was defined using a cutoff of 98%. Results: In 13 healthy donors, the mean percentage of ZAP-70 protein expression obtained by flow cytometry with unconjugated mAb (clone 2F3.2) was 4.69% ± 1.93 [range 2–9%] and the R ratio was 6.64 ± 1.54 and &gt; 4.8. In 83 B-CLL samples, ZAP-70 expression was determined using the same method and compared to IgVH mutational status. Results in table below show a 75% concordance between gene mutations and ZAP-70 expression when considering a percentage of positive B-cells &gt; 20%. A better concordance (81%) is obtained with a threshold T-cell MCF/ B-cell MCF at 4 determined by Youden’s index. To note the high concordance (90%) between unmutated status and ZAP-70 + expression (19/21). Comparison with at least 1 of the 3 conjugated mAbs has been performed for 63 samples, with discordant results in our laboratories. 62 mutated IgVH samples 21 unmutated IgVH samples ZAP-70 B-Cells + ≤ 20 % : 43 &gt; 20% : 19 T-cell MCF/B-cell MCF ≥ 4 : 48 &lt; 4 : 19 Conclusions: Our data document the concordance between IgVH gene mutational status and ZAP-70 protein expression measured by flow cytometry, particularly in ZAP-70 negative samples. We found that the indirect method of labelling with unconjugated anti-ZAP-70 mAb remains until now, in our hands, the gold standard method compared to the available dyes conjugate mAbs.

scholarly journals Cinnamic Acid (CINN) Induces Apoptosis and Proliferation in Human Nasopharyngeal Carcinoma Cells

2016 ◽  
Vol 40 (3-4) ◽  
pp. 589-596 ◽  
Author(s):  
Guangying Qi ◽  
Jian Chen ◽  
Changrong Shi ◽  
Yukun Wang ◽  
Sisi Mi ◽  
...  
Keyword(s):  
Protein Expression ◽  
Molecular Mechanisms ◽  
Flow Cytometric Analysis ◽  
D1 Protein ◽  
Inhibitory Effect ◽  
Flow Cytometric ◽  
Proliferation And Apoptosis ◽  
Dependent Flow ◽  
Fas L ◽  
Related Proteins

Background/Aims: CINN is the main ingredient of the traditional Chinese medicine cinnamon. The purpose of the present study was to investigate the effects of CINN on the proliferation and apoptosis of NPC cells and to elucidate the underlying molecular mechanisms. Materials and Methods: CNE2 human NPC cells were treated with various CINN concentrations. The effects of CINN on the proliferation and apoptosis of CNE2 NPC cells were examined using the MTT assay and flow cytometric analysis. Additionally, western blotting was performed to analyze the expression of a number of cell cycle- and apoptosis-related proteins. Results: The proliferation of CNE2 cells was significantly inhibited after treatment with different CINN concentrations for various lengths of time. The inhibitory effect of CINN was concentration-and time-dependent. Flow cytometric analysis showed that 2 mmol/L CINN displayed a significant apoptosis-inducing effect. The western blot analysis results showed that KLF6, Fas-L, Bax, P53 and caspase-3 protein expression was drastically increased in the CNE2 cells after treatment with 2 mmol/L CINN, whereas Bcl-2 and cyclin D1 protein expression was markedly reduced. Conclusion: CINN inhibits the proliferation and induces the apoptosis of CNE2 cells. Therefore, CINN possesses a potential anti-tumor effect.

scholarly journals Lactate dilates cochlear capillaries via type V fibrocyte-vessel coupling signaled by nNOS

2011 ◽  
Vol 301 (4) ◽  
pp. H1248-H1254 ◽  
Author(s):  
Min Dai ◽  
Yue Yang ◽  
Xiaorui Shi
Keyword(s):  
Blood Flow ◽  
Inner Ear ◽  
Regional Blood Flow ◽  
High Energy ◽  
No Synthase ◽  
Monocarboxylate Transporter ◽  
No Production ◽  
Spiral Ligament ◽  
Monocarboxylate Transporter 1 ◽  
Type V

Transduction of sound in the inner ear demands tight control over delivery of oxygen and glucose. However, the mechanisms underlying the control of regional blood flow are not yet fully understood. In this study, we report a novel local control mechanism that regulates cochlear blood flow to the stria vascularis, a high energy-consuming region of the inner ear. We found that extracellular lactate had a vasodilatory effect on the capillaries of the spiral ligament under both in vitro and in vivo conditions. The lactate, acting through monocarboxylate transporter 1 (MCT1), initiated neuronal nitric oxide (NO) synthase (nNOS) and catalyzed production of NO for the vasodilation. Blocking MCT1 with the MCT blocker, α-cyano-4-hydroxycinnamate (CHC), or a suppressing NO production with either the nonspecific inhibitor of NO synthase, NG-nitro-l-arginine methyl ester (l-NAME), or either of two selective nNOS inhibitors, 3-bromo-7-nitroindazole or (4S)- N-(4-amino-5[aminoethyl]aminopentyl)- N′-nitroguanidine (TFA), totally abolished the lactate-induced vasodilation. Pretreatment with the selective endothelial NO synthase inhibitor, l- N5-(1-iminoethyl)ornithine (l-NIO), eliminated the inhibition of lactate-induced vessel dilation. With immunohistochemical labeling, we found the expression of MCT1 and nNOS in capillary-coupled type V fibrocytes. The data suggest that type V fibrocytes are the source of the lactate-induced NO. Cochlear microvessel tone, regulated by lactate, is mediated by an NO-signaled coupling of fibrocytes and capillaries.

The Role of Beclin1 Gene in Autophagy and Apoptosis Induced by Ionizing Radiation in MCF - 7 Cells

2013 ◽  
Vol 790 ◽  
pp. 587-589
Author(s):  
Ya Li Qi ◽  
Yan Jun Liu ◽  
Da Li Zhao
Keyword(s):  
Ionizing Radiation ◽  
Protein Expression ◽  
Flow Cytometric Analysis ◽  
Beclin 1 ◽  
Over Expression ◽  
Western Blot Method ◽  
Flow Cytometric ◽  
Dose Effects ◽  
Mcf 7

Objective To discuss the role of Beclin1 gene in autophagy and apoptosis induced by ionizing radiation in MCF - 7 cells. Methods MTT assay was used to detect the influence of cells proliferation when beclin-1 gene was over expression and interference. Flow cytometric analysis detected the change of MCF - 7 cells apoptosis after irradiating by X-ray. Western blot method detected total protein changes of beclin - 1. Results Beclin-1 gene was interferred partly, the amount of protein expression of MCF-7-beclin1Ri cells reached to the lowest at 4 h, rose at 8 h, got to the most at 16 h and decreased at 32 h, but still held at higher level;Beclin-1 gene over-expression, the amount of protein expression increased gradually with the extension of time, and got to peak at 32 h. Conclusions Ionizing radiation can stimulate beclin-1 and exist relations of dose - effects in MCF-7 cells.

scholarly journals Publishing flow cytometry data

2010 ◽  
Vol 298 (2) ◽  
pp. L127-L130 ◽  
Author(s):  
D. F. Alvarez ◽  
K. Helm ◽  
J. DeGregori ◽  
M. Roederer ◽  
S. Majka
Keyword(s):  
Flow Cytometry ◽  
Protein Expression ◽  
Flow Cytometric Analysis ◽  
Experimental Information ◽  
Complex Data ◽  
Flow Cytometry Data ◽  
Flow Cytometric Data ◽  
Minimum Standards ◽  
Flow Cytometric ◽  
Cellular Measurements

Cellular measurements by flow cytometric analysis constitute an important step toward understanding individual attributes within a population of cells. Assessing individual cells within a population by protein expression using fluorescently labeled antibodies and other fluorescent probes can identify cellular patterns. The technology for accurately identifying subtle changes in protein expression within a population of cells using a vast array of technology has resulted in controversy and questions regarding reproducibility, which can be explained at least in part by the absence of standard methods to facilitate comparison of flow cytometric data. The complexity of technological advancements and the need for improvements in biological resolution results in the generation of complex data that demands the use of minimum standards for their publication. Herein we present a summarized view for the inclusion of consistent flow cytometric experimental information as supplemental data. Four major points, experimental and sample information, data acquisition, analysis, and presentation are emphasized. Together, these guidelines will facilitate the review and publication of flow cytometry data that provide an accurate foundation for ongoing studies with this evolving technology.

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