113 EXPRESSION OF BETA-OXIDATION-RELATED GENES IN PREECLAMPSIA-LIKE MODEL UNDER HYPOXIC CONDITION IN VIVO AND IN VITRO

2016 ◽  
Vol 28 (2) ◽  
pp. 187
Author(s):  
M. H. Lee ◽  
E.-K. Shin ◽  
H. Y. Kang ◽  
J.-U. Hwang ◽  
E.-B. Jeung
Keyword(s):  
Hypoxic Condition ◽  
Hypoxic Stress ◽  
Beta Oxidation ◽  
Experimental Conditions ◽  
Peripheral Tissues ◽  
Bewo Cells ◽  
In Vivo Experiments ◽  
Mouse Placenta

Preeclampsia (PE) is a disorder of pregnancy characterized by high blood pressure and large amounts of protein in the urine. Preeclampsia is thought in many cases to be caused by a shallowly implanted placenta that becomes hypoxic. The hypoxic condition during the pregnancy can result from a failure at any stage in the delivery of oxygen to the cells. In peripheral tissues, oxygen diffuses down a pressure gradient into cells and moves into their mitochondria, where it is used to produce energy. As an expression of beta-oxidation-related genes, ACADVL was detected by gene-fishing technology using the placenta of human. We conducted in vitro and in vivo experiments to confirm preliminary study by inducing hypoxic stress in the BeWo cells and mice placenta. BeWo cells were cultured at 37°C under 1% O2, 5% CO2, and balanced with N2. Pregnant mice were maintained from GD 6.5 to 17.5 under 11% O2, 5% CO2, and balanced with N2. The expression of beta-oxidation related genes (ACADVL, EHHADH, HADH, ACAA1) were observed under hypoxic condition at mRNA and protein levels. The expression of genes known as biomarkers for hypoxia, HIF-1α, was increased in BeWo cells and mouse placenta, which induced PE. The beta-oxidation-related genes ACADVL expression was significantly increased by hypoxic stress both BeWo cells and mouse placenta. The elevated level of HIF-1α indicates that our experimental conditions closely mimicked PE. These results indicate that changes of beta-oxidation-related genes are correlated with PE induced hypoxic condition.

217 THE EXPRESSION OF β-OXIDATION RELATING GENES UNDER HYPOXIC STRESS IN HUMAN PLACENTAL CHORIOCARCINOMA CELL (BeWo)

2015 ◽  
Vol 27 (1) ◽  
pp. 198
Author(s):  
E.-K. Shin ◽  
E.-B. Jeung
Keyword(s):  
Hypoxic Condition ◽  
Bewo Cell ◽  
Hypoxic Stress ◽  
Experimental Conditions ◽  
Fluorescent Intensity ◽  
Peripheral Tissues ◽  
Bewo Cells ◽  
Normoxic Control ◽  
La Jolla

Preeclampsia (PE) is thought in many cases to be caused by a shallowly implanted placenta that becomes hypoxic. Hypoxia can result from a failure at any stage in the delivery of oxygen to the cells. In peripheral tissues, oxygen diffuses down a pressure gradient into cells and moves into their mitochondria, where it is used to produce energy in conjunction with the breakdown of glucose, fats, and some amino acids. The aim of the study was to investigate the question of whether hypoxic stress is involved in β-oxidation of human placental BeWo cells. One of the β-oxidation related genes, ACADVL was detected by gene-fishing technology using the preeclamptic placenta of human. We conducted in vitro experiments to confirm a preliminary study by inducing hypoxic stress in the human placental BeWo cell. BeWo cells were cultured at 37°C in a 20% O2, 5% CO2 humidified tissue culture incubator. And then we induced hypoxic stress in BeWo cell cultured under 1% O2, 5% CO2, and balanced with N2. The BeWo cells were divided into three groups: normoxia, hypoxia 24 h, and hypoxia 48 h. The expression of β-oxidation related genes (ACADVL, EHHADH, HADH, ACAA) were observed under hypoxic condition in BeWo cells by using real-time RT-PCR. Relative quantification with HPRT1 was based on the comparison of CT at a constant fluorescent intensity. The amount of transcript is inversely related to the observed CT, and for every 2-fold dilution in the transcript, CT is expected to increase by 1. Relative expression was calculated using the equation R = 2–(CTsample – CTcontrol). Data were analysed with a nonparametric one-way ANOVA, followed by Tukey's test for multiple comparisons. All statistical analyses were performed using GraphPad Prism software (v 4.0, GraphPad Software, La Jolla, CA, USA). P-values <0.05 were considered statistically significant. The expression of a gene known as a biomarker for hypoxia, HIF-1a, was significantly increased in BeWo cells which induced preeclampsia. The elevated level of HIF-1a indicates that our experimental conditions closely mimicked preeclampsia. The β-oxidation related genes, ACADVL, EHHADH, and HADH expressions were significantly increased under hypoxic condition in BeWo compared with normoxic control. These results indicate that changes of β-oxidation related genes observed under hypoxic BeWo cells are similar to ones associated with preeclampsia, and the expression of β-oxidation related genes were up-regulated by hypoxic stress. They might be involved in pathogenesis of preeclampsia during gestation. Taken together, increase of β-oxidation-related genes under hypoxic stress may cause a compensation of energy metabolism deficiency through lipid metabolism.

Toxicity of ligand-dependent Cre recombinases and generation of a conditional Cre deleter mouse allowing mosaic recombination in peripheral tissues

2007 ◽  
Vol 31 (1) ◽  
pp. 32-41 ◽  
Author(s):  
Dorothe Hameyer ◽  
Ate Loonstra ◽  
Leonid Eshkind ◽  
Steffen Schmitt ◽  
Cecilia Antunes ◽  
...  
Keyword(s):  
Gene Function ◽  
Background Activity ◽  
Es Cells ◽  
Embryonic Stem ◽  
Coding Region ◽  
Peripheral Tissues ◽  
Rosa26 Locus ◽  
In Vivo Experiments

Ligand-activated Cre recombinases are widely used for studying gene function in vitro and in conditional mouse models. To compare ligand-dependent Cre recombinases, different Cre estrogen receptor fusions were introduced into the ROSA26 locus of embryonic stem (ES) cells and assayed for genotoxicity and recombination efficiency. Of the tested recombinases, the CreERT2 variant showed no toxicity and was highly responsive to ligand induction. To constitutively express CreERT2 in mice and also to clarify whether the CreERT2 system displays background activity, we generated a knock-in mouse line harboring the CreERT2 coding region under the control of the ROSA26 locus. Analysis of this ROSA26-CreERT2 deleter mouse with different reporter strains revealed ubiquitous recombination in the embryo and partial recombination in peripheral and hematopoietic tissues but no effective CreERT2 expression in the brain. Furthermore, using flow cytometry, we found low-level background recombination in noninduced bitransgenic ROSA26-CreERT2/EGFP reporter mice. To determine whether background activity poses a general problem for conducting conditional in vivo experiments with the ROSA26-CreERT2 deleter, we used a sensitive conditional skin cancer model. In this assay, cancer induction was completely restricted to induced bitransgenic CreERT2/K-RasV12 mice, whereas noninduced control animals did not show any sign of cancer, indicating the usefulness of the ROSA-CreERT2 system for regulating conditional gene expression in vivo. The ROSA26-CreERT2 deleter strain will be a convenient experimental tool for studying gene function under circumstances requiring partial induction of recombination in peripheral tissues and will be useful for uncovering previously unknown or unsuspected phenotypes.

scholarly journals FSMP-14. METABOLIC SYMBIOSIS IN GLIOBLASTOMA: LACTATE DEHYDROGENASES TAKE THE LEAD

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. i19-i19
Author(s):  
Joris Guyon ◽  
Irati Romero-Garmendia ◽  
Andreas Bikfalvi ◽  
Thomas Daubon
Keyword(s):  
Cell Invasion ◽  
Critical Role ◽  
Krebs Cycle ◽  
Lactate Production ◽  
Hypoxic Condition ◽  
Production Flow ◽  
In Vivo Experiments ◽  
Cell Energy

Abstract Glioblastoma (GBM) is a common and devastating brain tumor, associated with a low median survival, despite standard therapeutic management. Among its major features, GBMs are highly angiogenic, infiltrative, and exhibit paradoxically an elevated glycolysis. Most of differentiated cells convert glucose into pyruvate that enters into the Krebs cycle to maximize energy production in the presence of oxygen. For cancer cells, glucose uptake and catabolism are increased regardless of oxygen level. However, tumor cell energy needs are important, mainly for rapid growth, which require a much faster production flow. Lactate dehydrogenase (LDH) are involved at this step. LDHA converts pyruvate into lactate, and generates NAD+ to maintain glycolysis. Thus, lactate is exported into the extracellular compartment inducing an acidification of the microenvironment. Moreover, LDHB, another LDH isoform, metabolizes lactate into pyruvate for generating energy in mitochondria. LDHB is generally expressed in oligodendrocytes or neurons, but also in GBM cells. Though LDHA has already been studied in many cancers including GBM, the simultaneous role of LDH enzymes have not yet been investigated in GBM development. Our results showed that hypoxia significantly increased LDHA expression and lactate production, but no changes were observed for LDHB. In presence of lactate, cell invasion was significantly increased. In vitro results showed that, under hypoxic condition, double sgLDHA/B cell growth and invasion was dramatically decreased in comparison to control cells, mainly caused by an increase in apoptosis. In vivo experiments showed that double impairment of LDHA and B significantly reduced tumor growth and cell invasion, and induces a massive increase in mouse survival. Considered for a long time as a metabolic waste, lactate is shown here to play a critical role in the tumor niche. This study highlighted GBM adaptability through the LDH isoforms and their involvement in GBM development.

scholarly journals Effects of Endogenous d-Alanine Synthesis and Autoinhibition of Bacillus anthracis Germination on In Vitro and In Vivo Infections

2007 ◽  
Vol 75 (12) ◽  
pp. 5726-5734 ◽  
Author(s):  
Matthew T. McKevitt ◽  
Katie M. Bryant ◽  
Salika M. Shakir ◽  
Jason L. Larabee ◽  
Steven R. Blanke ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Lethal Dose ◽  
Threshold Effect ◽  
Experimental Conditions ◽  
In Vivo Experiments ◽  
Endogenous Production ◽  
Dormant Spore ◽  
Alanine Production

ABSTRACT Bacillus anthracis transitions from a dormant spore to a vegetative bacillus through a series of structural and biochemical changes collectively referred to as germination. The timing of germination is important during early steps in infection and may determine if B. anthracis survives or succumbs to responsive macrophages. In the current study experiments determined the contribution of endogenous d-alanine production to the efficiency and timing of B. anthracis spore germination under in vitro and in vivo conditions. Racemase-mediated production of endogenous d-alanine by B. anthracis altered the kinetics for initiation of germination over a range of spore densities and exhibited a threshold effect wherein small changes in spore number resulted in major changes in germination efficiency. This threshold effect correlated with d-alanine production, was prevented by an alanine racemase inhibitor, and required l-alanine. Interestingly, endogenous production of inhibitory levels of d-alanine was detected under experimental conditions that did not support germination and in a germination-deficient mutant of B. anthracis. Racemase-dependent production of d-alanine enhanced survival of B. anthracis during interaction with murine macrophages, suggesting a role for inhibition of germination during interaction with these cells. Finally, in vivo experiments revealed an approximately twofold decrease in the 50% lethal dose of B. anthracis spores administered in the presence of d-alanine, indicating that rates of germination may be directly influenced by the levels of this amino acid during early stages of disease.

Superoxide, Xanthine Oxidase and Platelet Reactions: Further Studies on Mechanisms by which Oxidants Influence Platelets

1981 ◽  
Vol 45 (03) ◽  
pp. 290-293 ◽  
Author(s):  
Peter H Levine ◽  
Danielle G Sladdin ◽  
Norman I Krinsky
Keyword(s):  
Superoxide Dismutase ◽  
Cytochrome C ◽  
Xanthine Oxidase ◽  
Direct Effect ◽  
Platelet Function ◽  
Experimental Conditions ◽  
Release Reaction

SummaryIn the course of studying the effects on platelets of the oxidant species superoxide (O- 2), Of was generated by the interaction of xanthine oxidase plus xanthine. Surprisingly, gel-filtered platelets, when exposed to xanthine oxidase in the absence of xanthine substrate, were found to generate superoxide (O- 2), as determined by the reduction of added cytochrome c and by the inhibition of this reduction in the presence of superoxide dismutase.In addition to generating Of, the xanthine oxidase-treated platelets display both aggregation and evidence of the release reaction. This xanthine oxidase induced aggreagtion is not inhibited by the addition of either superoxide dismutase or cytochrome c, suggesting that it is due to either a further metabolite of O- 2, or that O- 2 itself exerts no important direct effect on platelet function under these experimental conditions. The ability of Of to modulate platelet reactions in vivo or in vitro remains in doubt, and xanthine oxidase is an unsuitable source of O- 2 in platelet studies because of its own effects on platelets.

Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

1997 ◽  
Vol 77 (05) ◽  
pp. 0975-0980 ◽  
Author(s):  
Angel Gálvez ◽  
Goretti Gómez-Ortiz ◽  
Maribel Díaz-Ricart ◽  
Ginés Escolar ◽  
Rogelio González-Sarmiento ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Platelet Adhesion ◽  
Umbilical Vein ◽  
Procoagulant Activity ◽  
Experimental Conditions ◽  
Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

ANTITUMOR EFFECT OF COLLOIDAL SILVER BISILICATE IN EXPERIMENTAL IN VITRO AND IN VIVO STUDIES

2019 ◽  
Vol 65 (5) ◽  
pp. 760-765
Author(s):  
Margarita Tyndyk ◽  
Irina Popovich ◽  
A. Malek ◽  
R. Samsonov ◽  
N. Germanov ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Human Tumor ◽  
Significant Inhibition ◽  
In Vivo Studies ◽  
Ehrlich Carcinoma ◽  
In Vivo Experiments

The paper presents the results of the research on the antitumor activity of a new drug - atomic clusters of silver (ACS), the colloidal solution of nanostructured silver bisilicate Ag6Si2O7 with particles size of 1-2 nm in deionized water. In vitro studies to evaluate the effect of various ACS concentrations in human tumor cells cultures (breast cancer, colon carcinoma and prostate cancer) were conducted. The highest antitumor activity of ACS was observed in dilutions from 2.7 mg/l to 5.1 mg/l, resulting in the death of tumor cells in all studied cell cultures. In vivo experiments on transplanted Ehrlich carcinoma model in mice consuming 0.75 mg/kg ACS with drinking water revealed significant inhibition of tumor growth since the 14th day of experiment (maximally by 52% on the 28th day, p < 0.05) in comparison with control. Subcutaneous injections of 2.5 mg/kg ACS inhibited Ehrlich's tumor growth on the 7th and 10th days of the experiment (p < 0.05) as compared to control.

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

scholarly journals Non-invasive skin sampling of tryptophan/kynurenine ratio in vitro towards a skin cancer biomarker

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Skaidre Jankovskaja ◽  
Johan Engblom ◽  
Melinda Rezeli ◽  
György Marko-Varga ◽  
Tautgirdas Ruzgas ◽  
...  
Keyword(s):  
Skin Cancer ◽  
Relative Increase ◽  
Cancer Biomarker ◽  
Cancer Diagnostics ◽  
Sampling Time ◽  
Skin Permeability ◽  
Experimental Conditions ◽  
Non Invasive

AbstractThe tryptophan to kynurenine ratio (Trp/Kyn) has been proposed as a cancer biomarker. Non-invasive topical sampling of Trp/Kyn can therefore serve as a promising concept for skin cancer diagnostics. By performing in vitro pig skin permeability studies, we conclude that non-invasive topical sampling of Trp and Kyn is feasible. We explore the influence of different experimental conditions, which are relevant for the clinical in vivo setting, such as pH variations, sampling time, and microbial degradation of Trp and Kyn. The permeabilities of Trp and Kyn are overall similar. However, the permeated Trp/Kyn ratio is generally higher than unity due to endogenous Trp, which should be taken into account to obtain a non-biased Trp/Kyn ratio accurately reflecting systemic concentrations. Additionally, prolonged sampling time is associated with bacterial Trp and Kyn degradation and should be considered in a clinical setting. Finally, the experimental results are supported by the four permeation pathways model, predicting that the hydrophilic Trp and Kyn molecules mainly permeate through lipid defects (i.e., the porous pathway). However, the hydrophobic indole ring of Trp is suggested to result in a small but noticeable relative increase of Trp diffusion via pathways across the SC lipid lamellae, while the shunt pathway is proposed to slightly favor permeation of Kyn relative to Trp.

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