scholarly journals Multiple Factors Affecting Cellular Redox Status and Energy Metabolism Modulate Hypoxia-Inducible Factor Prolyl Hydroxylase Activity In Vivo and In Vitro

2006 ◽  
Vol 27 (3) ◽  
pp. 912-925 ◽  
Author(s):  
Yi Pan ◽  
Kyle D. Mansfield ◽  
Cara C. Bertozzi ◽  
Viktoriya Rudenko ◽  
Denise A. Chan ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Hypoxia Inducible Factor ◽  
Simultaneous Detection ◽  
Tca Cycle ◽  
Cellular Responses ◽  
Prolyl Hydroxylase ◽  
In Vitro Assays ◽  
Tca Cycle Intermediates

ABSTRACT Prolyl hydroxylation of hypoxible-inducible factor alpha (HIF-α) proteins is essential for their recognition by pVHL containing ubiquitin ligase complexes and subsequent degradation in oxygen (O2)-replete cells. Therefore, HIF prolyl hydroxylase (PHD) enzymatic activity is critical for the regulation of cellular responses to O2 deprivation (hypoxia). Using a fusion protein containing the human HIF-1α O2-dependent degradation domain (ODD), we monitored PHD activity both in vivo and in cell-free systems. This novel assay allows the simultaneous detection of both hydroxylated and nonhydroxylated PHD substrates in cells and during in vitro reactions. Importantly, the ODD fusion protein is regulated with kinetics identical to endogenous HIF-1α during cellular hypoxia and reoxygenation. Using in vitro assays, we demonstrated that the levels of iron (Fe), ascorbate, and various tricarboxylic acid (TCA) cycle intermediates affect PHD activity. The intracellular levels of these factors also modulate PHD function and HIF-1α accumulation in vivo. Furthermore, cells treated with mitochondrial inhibitors, such as rotenone and myxothiazol, provided direct evidence that PHDs remain active in hypoxic cells lacking functional mitochondria. Our results suggest that multiple mitochondrial products, including TCA cycle intermediates and reactive oxygen species, can coordinate PHD activity, HIF stabilization, and cellular responses to O2 depletion.

scholarly journals HIF-1α is a key mediator of the lung inflammatory potential of lithium-ion battery particles

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Violaine Sironval ◽  
Mihaly Palmai-Pallag ◽  
Rita Vanbever ◽  
François Huaux ◽  
Jorge Mejia ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Hypoxia Inducible Factor ◽  
Lithium Ion ◽  
In Vitro Assays ◽  
Inhalation Toxicity ◽  
Cobalt Nickel ◽  
Occupational Settings

Abstract Background Li-ion batteries (LIB) are increasingly used worldwide. They are made of low solubility micrometric particles, implying a potential for inhalation toxicity in occupational settings and possibly for consumers. LiCoO2 (LCO), one of the most used cathode material, induces inflammatory and fibrotic lung responses in mice. LCO also stabilizes hypoxia-inducible factor (HIF) -1α, a factor implicated in inflammation, fibrosis and carcinogenicity. Here, we investigated the role of cobalt, nickel and HIF-1α as determinants of toxicity, and evaluated their predictive value for the lung toxicity of LIB particles in in vitro assays. Results By testing a set of 5 selected LIB particles (LCO, LiNiMnCoO2, LiNiCoAlO2) with different cobalt and nickel contents, we found a positive correlation between their in vivo lung inflammatory activity, and (i) Co and Ni particle content and their bioaccessibility and (ii) the stabilization of HIF-1α in the lung. Inhibition of HIF-1α with chetomin or PX-478 blunted the lung inflammatory response to LCO in mice. In IL-1β deficient mice, HIF-1α was the upstream signal of the inflammatory lung response to LCO. In vitro, the level of HIF-1α stabilization induced by LIB particles in BEAS-2B cells correlated with the intensity of lung inflammation induced by the same particles in vivo. Conclusions We conclude that HIF-1α, stabilized in lung cells by released Co and Ni ions, is a mechanism-based biomarker of lung inflammatory responses induced by LIB particles containing Co/Ni. Documenting the Co/Ni content of LIB particles, their bioaccessibility and their capacity to stabilize HIF-1α in vitro can be used to predict the lung inflammatory potential of LIB particles.

scholarly journals Cystathione β-synthase regulates HIF-1α stability through persulfidation of PHD2

2020 ◽  
Vol 6 (27) ◽  
pp. eaaz8534
Author(s):  
Anindya Dey ◽  
Shubhangi Prabhudesai ◽  
Yushan Zhang ◽  
Geeta Rao ◽  
Karthikeyan Thirugnanam ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Hypoxia Inducible Factor ◽  
Point Mutations ◽  
Prolyl Hydroxylase ◽  
Zinc Finger Motif ◽  
Hydroxylase Activity ◽  
Zebrafish Model ◽  
Enzymatic Action

The stringent expression of the hypoxia inducible factor-1α (HIF-1α) is critical to a variety of pathophysiological conditions. We reveal that, in normoxia, enzymatic action of cystathionine β-synthase (CBS) produces H2S, which persulfidates prolyl hydroxylase 2 (PHD2) at residues Cys21 and Cys33 (zinc finger motif), augmenting prolyl hydroxylase activity. Depleting endogenous H2S either by hypoxia or by inhibiting CBS via chemical or genetic means reduces persulfidation of PHD2 and inhibits activity, preventing hydroxylation of HIF-1α, resulting in stabilization. Our in vitro findings are further supported by the depletion of CBS in the zebrafish model that exhibits axis defects and abnormal intersegmental vessels. Exogenous H2S supplementation rescues both in vitro and in vivo phenotypes. We have identified the persulfidated residues and defined their functional significance in regulating the activity of PHD2 via point mutations. Thus, the CBS/H2S/PHD2 axis may provide therapeutic opportunities for pathologies associated with HIF-1α dysregulation in chronic diseases.

scholarly journals Development of a LC-MS/MS Method for the Simultaneous Detection of Tricarboxylic Acid Cycle Intermediates in a Range of Biological Matrices

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Omar Al Kadhi ◽  
Antonietta Melchini ◽  
Richard Mithen ◽  
Shikha Saha
Keyword(s):  
Ex Vivo ◽  
Tricarboxylic Acid Cycle ◽  
Simultaneous Detection ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
In Vivo Studies ◽  
Biological Matrices ◽  
Wide Range ◽  
Tca Cycle Intermediates

It is now well-established that perturbations in the tricarboxylic acid (TCA) cycle play an important role in the metabolic transformation occurring in cancer including that of the prostate. A method for simultaneous qualitative and quantitative analysis of TCA cycle intermediates in body fluids, tissues, and cultured cell lines of human origin was developed using a common C18 reversed-phase column by LC-MS/MS technique. This LC-MS/MS method for profiling TCA cycle intermediates offers significant advantages including simple and fast preparation of a wide range of human biological samples. The analytical method was validated according to the guideline of the Royal Society of Chemistry Analytical Methods Committee. The limits of detection were below 60 nM for most of the TCA intermediates with the exception of lactic and fumaric acids. The calibration curves of all TCA analytes showed linearity with correlation coefficientsr2>0.9998. Recoveries were >95% for all TCA analytes. This method was established taking into consideration problems and limitations of existing techniques. We envisage that its application to different biological matrices will facilitate deeper understanding of the metabolic changes in the TCA cycle from in vitro, ex vivo, and in vivo studies.

scholarly journals The Zinc Finger of Prolyl Hydroxylase Domain Protein 2 Is Essential for Efficient Hydroxylation of Hypoxia-Inducible Factor α

2016 ◽  
Vol 36 (18) ◽  
pp. 2328-2343 ◽  
Author(s):  
Patrick R. Arsenault ◽  
Daisheng Song ◽  
Yu Jin Chung ◽  
Tejvir S. Khurana ◽  
Frank S. Lee
Keyword(s):  
High Altitude ◽  
Zinc Finger ◽  
Hypoxia Inducible Factor ◽  
Prolyl Hydroxylase ◽  
Loss Of Function ◽  
Domain Protein ◽  
Factor Α ◽  
Prolyl Hydroxylase Domain

Prolyl hydroxylase domain protein 2 (PHD2) (also known as EGLN1) is a key oxygen sensor in mammals that posttranslationally modifies hypoxia-inducible factor α (HIF-α) and targets it for degradation. In addition to its catalytic domain, PHD2 contains an evolutionarily conserved zinc finger domain, which we have previously proposed recruits PHD2 to the HSP90 pathway to promote HIF-α hydroxylation. Here, we provide evidence that this recruitment is critical bothin vitroandin vivo. We show thatin vitro, the zinc finger can function as an autonomous recruitment domain to facilitate interaction with HIF-α.In vivo, ablation of zinc finger function by a C36S/C42SEgln1knock-in mutation results in upregulation of theerythropoietingene, erythrocytosis, and augmented hypoxic ventilatory response, all hallmarks ofEgln1loss of function and HIF stabilization. Hence, the zinc finger ordinarily performs a critical positive regulatory function. Intriguingly, the function of this zinc finger is impaired in high-altitude-adapted Tibetans, suggesting that their adaptation to high altitude may, in part, be due to a loss-of-functionEGLN1allele. Thus, these findings have important implications for understanding both the molecular mechanism of the hypoxic response and human adaptation to high altitude.

scholarly journals A Feedback Loop Involving the Phd3 Prolyl Hydroxylase Tunes the Mammalian Hypoxic Response In Vivo

2009 ◽  
Vol 29 (21) ◽  
pp. 5729-5741 ◽  
Author(s):  
Yoji Andrew Minamishima ◽  
Javid Moslehi ◽  
Robert F. Padera ◽  
Roderick T. Bronson ◽  
Ronglih Liao ◽  
...  
Keyword(s):  
Feedback Loop ◽  
Target Genes ◽  
Hypoxia Inducible Factor ◽  
Premature Mortality ◽  
Prolyl Hydroxylase ◽  
Α Subunit ◽  
Von Hippel Lindau ◽  
Regulatory Feedback Loop

ABSTRACT Hypoxia-inducible factor (HIF), consisting of a labile α subunit and a stable β subunit, is a master regulator of hypoxia-responsive mRNAs. HIFα undergoes oxygen-dependent prolyl hydroxylation, which marks it for polyubiquitination by a complex containing the von Hippel-Lindau protein (pVHL). Among the three Phd family members, Phd2 appears to be the primary HIF prolyl hydroxylase. Phd3 is induced by HIF and, based on findings from in vitro studies, may participate in a HIF-regulatory feedback loop. Here, we report that Phd3 loss exacerbates the HIF activation, hepatic steatosis, dilated cardiomyopathy, and premature mortality observed in mice lacking Phd2 alone and produces a closer phenocopy of the changes seen in mice lacking pVHL than the loss of Phd2 alone. Importantly, the degree to which Phd3 can compensate for Phd2 loss and the degree to which the combined loss of Phd2 and Phd3 resembles pVHL loss appear to differ for different HIF-responsive genes and in different tissues. These findings highlight that the responses of different HIF target genes to changes in prolyl hydroxylase activity differ, quantitatively and qualitatively, in vivo and have implications for the development of paralog-specific prolyl hydroxylase inhibitors as therapeutic agents.

scholarly journals Pseudomonas expression of an oxygen sensing prolyl hydroxylase homologue regulates neutrophil host responses in vitro and in vivo

2017 ◽  
Vol 2 ◽  
pp. 104 ◽  
Author(s):  
Rebecca S. Dickinson ◽  
Fiona Murphy ◽  
Catherine Doherty ◽  
Sam Williams ◽  
Ananda Mirchandani ◽  
...  
Keyword(s):  
Active Sites ◽  
Hypoxia Inducible Factor ◽  
Elongation Factor ◽  
Prolyl Hydroxylase ◽  
Human Neutrophils ◽  
Neutrophil Apoptosis ◽  
Wild Type ◽  
Prolyl Hydroxylases

Background: Pseudomonas species are adapted to evade innate immune responses and can persist at sites of relative tissue hypoxia, including the mucus-plugged airways of patients with cystic fibrosis and bronchiectasis.  The ability of these bacteria to directly sense and respond to changes in local oxygen availability is in part consequent upon expression of the 2-oxoglutarate oxygenase, Pseudomonas prolyl hydroxylase (PPHD), which acts on elongation factor Tu (EF-Tu), and is homologous with the human hypoxia inducible factor (HIF) prolyl hydroxylases. We report that PPHD expression regulates the neutrophil response to acute pseudomonal infection. Methods: In vitro co-culture experiments were performed with human neutrophils and PPHD-deficient and wild-type bacteria and supernatants, with viable neutrophil counts determined by flow cytometry. In vivo consequences of infection with PPHD deficient P. aeruginosa were determined in an acute pneumonia mouse model following intra-tracheal challenge. Results: Supernatants of PPHD-deficient bacterial cultures contained higher concentrations of the phenazine exotoxin pyocyanin and induced greater acceleration of neutrophil apoptosis than wild-type PAO1 supernatants in vitro.  In vivo infection with PPHD mutants compared to wild-type PAO1 controls resulted in increased levels of neutrophil apoptosis and impaired control of infection, with higher numbers of P. aeruginosa recovered from the lungs of mice infected with the PPHD-deficient strain.  This resulted in an overall increase in mortality in mice infected with the PPHD-deficient strain. Conclusions: Our data show that Pseudomonas expression of its prolyl hydroxylase influences the outcome of host-pathogen interactions in vitro and in vivo, demonstrating the importance of considering how both host and pathogen adaptations to hypoxia together define outcomes of infection. Given that inhibitors for the HIF prolyl hydroxylases are in late stage trials for the treatment of anaemia and that the active sites of PPHD and human HIF prolyl hydroxylases are closely related, the results are of current clinical interest.

A in Vivo Assay for Standardizing Heparin Preparations

1979 ◽  
Vol 41 (03) ◽  
pp. 576-582
Author(s):  
A R Pomeroy
Keyword(s):  
In Vitro Assays ◽  
British Pharmacopoeia ◽  
In Vitro Method ◽  
Standard Preparation ◽  
Reliable Estimation ◽  
Assay Procedure ◽  
Accuracy And Precision ◽  
Heparin Preparation

SummaryThe limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use.The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a “2 and 2 dose assay” procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation.The similarity of results obtained the in vivo method and the in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.

Potentially Thrombogenic Materials in Factor IX Concentrates

1975 ◽  
Vol 33 (03) ◽  
pp. 617-631 ◽  
Author(s):  
H. S Kingdon ◽  
R. L Lundblad ◽  
J. J Veltkamp ◽  
D. L Aronson
Keyword(s):  
Human Plasma ◽  
Antithrombin Iii ◽  
Factor Ix ◽  
In Vitro Assays ◽  
Substantial Agreement ◽  
Coefficient Of Correlation

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.

In-silico Studies of Isolated Phytoalkaloid Against Lipoxygenase: Study Based on Possible Correlation

2018 ◽  
Vol 21 (3) ◽  
pp. 215-221
Author(s):  
Haroon Khan ◽  
Muhammad Zafar ◽  
Helena Den-Haan ◽  
Horacio Perez-Sanchez ◽  
Mohammad Amjad Kamal
Keyword(s):  
In Silico ◽  
Docking Studies ◽  
In Vivo Studies ◽  
In Vitro Assays ◽  
Chronic Obstructive ◽  
Lipoxygenase Inhibitors ◽  
Lipoxygenase Inhibition ◽  
In Silico Studies

Aim and Objective: Lipoxygenase (LOX) enzymes play an important role in the pathophysiology of several inflammatory and allergic diseases including bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, rheumatoid arthritis and chronic obstructive pulmonary disease. Inhibitors of the LOX are believed to be an ideal approach in the treatment of diseases caused by its over-expression. In this regard, several synthetic and natural agents are under investigation worldwide. Alkaloids are the most thoroughly investigated class of natural compounds with outstanding past in clinically useful drugs. In this article, we have discussed various alkaloids of plant origin that have already shown lipoxygenase inhibition in-vitro with possible correlation in in silico studies. Materials and Methods: Molecular docking studies were performed using MOE (Molecular Operating Environment) software. Among the ten reported LOX alkaloids inhibitors, derived from plant, compounds 4, 2, 3 and 1 showed excellent docking scores and receptor sensitivity. Result and Conclusion: These compounds already exhibited in vitro lipoxygenase inhibition and the MOE results strongly correlated with the experimental results. On the basis of these in vitro assays and computer aided results, we suggest that these compounds need further detail in vivo studies and clinical trial for the discovery of new more effective and safe lipoxygenase inhibitors. In conclusion, these results might be useful in the design of new and potential lipoxygenase (LOX) inhibitors.

Discovery of 3-Cinnamamido-N-Substituted Benzamides as Potential Antimalarial Agents

2020 ◽  
Vol 16 ◽  
Author(s):  
Haicheng Liu ◽  
Yushi Futamura ◽  
Honghai Wu ◽  
Aki Ishiyama ◽  
Taotao Zhang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Antimalarial Activity ◽  
In Vitro Assays ◽  
Compound Library ◽  
Antimalarial Agents ◽  
Phenotypic Screen ◽  
Ic50 Values ◽  
Substituted Benzamides

Background: Malaria is one of the most devastating parasitic diseases, yet the discovery of antimalarial agents remains profoundly challenging. Very few new antimalarials have been developed in the past 50 years, while the emergence of drug-resistance continues to appear. Objective: This study focuses on the discovery, design, synthesis, and antimalarial evaluation of 3-cinnamamido-N-substituted benzamides. Method: In this study, a screening of our compound library was carried out against the multidrug-sensitive Plasmodium falciparum 3D7 strain. Derivatives of the hit were designed, synthesized and tested against P. falciparum 3D7 and the in vivo antimalarial activity of the most active compounds was evaluated using the method of Peters’ 4-day suppressive test. Results: The retrieved hit compound 1 containing a 3-cinnamamido-N-substituted benzamide skeleton showed moderate antimalarial activity (IC50 = 1.20 µM) for the first time. A series of derivatives were then synthesized through a simple four-step workflow, and half of them exhibited slightly better antimalarial effect than the precursor 1 during the subsequent in vitro assays. Additionally, compounds 11, 23, 30 and 31 displayed potent activity with IC50 values of approximately 0.1 µM, and weak cytotoxicity against mammalian cells. However, in vivo antimalarial activity is not effective which might be ascribed to the poor solubility of these compounds. Conclusion: In this study, phenotypic screen of our compound library resulted in the first report of 3-cinnamamide framework with antimalarial activity and 40 derivatives were then designed and synthesized. Subsequent structure-activity studies showed that compounds 11, 23, 30 and 31 exhibited the most potent and selective activity against P. falciparum 3D7 strain with IC50 values around 0.1 µM. Our work herein sets another example of phenotypic screen-based drug discovery, leading to potentially promising candidates of novel antimalarial agents once given further optimization.

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