scholarly journals The radiosensitising effect of gemcitabine and its main metabolite dFdU under low oxygen conditions is in vitro not dependent on functional HIF-1 protein

BMC Cancer ◽  
2014 ◽  
Vol 14 (1) ◽  
Author(s):  
An Wouters ◽  
Bea Pauwels ◽  
Natalie Burrows ◽  
Marc Baay ◽  
Vanessa Deschoolmeester ◽  
...  
Keyword(s):  
Low Oxygen ◽  
Main Metabolite ◽  
Oxygen Conditions

scholarly journals Hypoxic Response of Mycobacterium tuberculosis Studied by Metabolic Labeling and Proteome Analysis of Cellular and Extracellular Proteins

2002 ◽  
Vol 184 (13) ◽  
pp. 3485-3491 ◽  
Author(s):  
Ida Rosenkrands ◽  
Richard A. Slayden ◽  
Janne Crawford ◽  
Claus Aagaard ◽  
Clifton E. Barry ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Proteome Analysis ◽  
Peptide Mass Fingerprinting ◽  
Extracellular Proteins ◽  
Metabolic Labeling ◽  
Low Oxygen ◽  
Hypoxic Response ◽  
Peptide Mass ◽  
Oxygen Conditions

ABSTRACT The events involved in the establishment of a latent infection with Mycobacterium tuberculosis are not fully understood, but hypoxic conditions are generally believed to be the environment encountered by the pathogen in the central part of the granuloma. The present study was undertaken to provide insight into M. tuberculosis protein expression in in vitro latency models where oxygen is depleted. The response of M. tuberculosis to low-oxygen conditions was investigated in both cellular and extracellular proteins by metabolic labeling, two-dimensional electrophoresis, and protein signature peptide analysis by liquid chromatography-mass spectrometry. By peptide mass fingerprinting and immunodetection, five proteins more abundant under low-oxygen conditions were identified from several lysates of M. tuberculosis: Rv0569, Rv2031c (HspX), Rv2623, Rv2626c, and Rv3841 (BfrB). In M. tuberculosis culture filtrates, two additional proteins, Rv0363c (Fba) and Rv2780 (Ald), were found in increased amounts under oxygen limitation. These results extend our understanding of the hypoxic response in M. tuberculosis and potentially provide important insights into the physiology of the latent bacilli.

scholarly journals Role of the rdxA and frxA genes in oxygen-dependent metronidazole resistance of Helicobacter pylori

2004 ◽  
Vol 53 (11) ◽  
pp. 1123-1128 ◽  
Author(s):  
Monique M Gerrits ◽  
Egbert-Jan van der Wouden ◽  
Dorine A Bax ◽  
Anton A van Zwet ◽  
Arnoud HM van Vliet ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Cell Viability ◽  
Protein Synthesis Inhibitor ◽  
Low Oxygen ◽  
Anaerobic Conditions ◽  
Metronidazole Resistance ◽  
Oxygen Conditions ◽  
H Pylori

Almost 50 % of all Helicobacter pylori isolates are resistant to metronidazole, which reduces the efficacy of metronidazole-containing regimens, but does not make them completely ineffective. This discrepancy between in vitro metronidazole resistance and treatment outcome may partially be explained by changes in oxygen pressure in the gastric environment, as metronidazole-resistant (MtzR) H. pylori isolates become metronidazole-susceptible (MtzS) under low oxygen conditions in vitro. In H. pylori the rdxA and frxA genes encode reductases which are required for the activation of metronidazole, and inactivation of these genes results in metronidazole resistance. Here the role of inactivating mutations in these genes on the reversibility of metronidazole resistance under low oxygen conditions is established. Clinical H. pylori isolates containing mutations resulting in a truncated RdxA and/or FrxA protein were selected and incubated under anaerobic conditions, and the effect of these conditions on the MICs of metronidazole, amoxycillin, clarithromycin and tetracycline, and cell viability were determined. While anaerobiosis had no effect on amoxycillin, clarithromycin and tetracycline resistance, all isolates lost their metronidazole resistance when cultured under anaerobic conditions. This loss of metronidazole resistance also occurred in the presence of the protein synthesis inhibitor chloramphenicol. Thus, factor(s) that activate metronidazole under low oxygen tension are not specifically induced by low oxygen conditions, but are already present under microaerophilic conditions. As there were no significant differences in cell viability between the clinical isolates, it is likely that neither the rdxA nor the frxA gene participates in the reversibility of metronidazole resistance.

scholarly journals Emergent Bacteria in Cystic Fibrosis:In VitroBiofilm Formation and Resilience under Variable Oxygen Conditions

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Susana P. Lopes ◽  
Nuno F. Azevedo ◽  
Maria O. Pereira
Keyword(s):  
Bacterial Species ◽  
Low Oxygen ◽  
Antibiotic Susceptibilities ◽  
Oxygen Conditions ◽  
Mode Of Life ◽  
Lung Infections ◽  
Biofilm Development ◽  
The Impact

Concurrent to conventional bacterial pathogens, unusual microbes are emerging from cystic fibrosis (CF) airways. Nonetheless, little is known about the contribution of these newly microbes to the resilience of CF-associated biofilms, particularly under variable-oxygen concentrations that are known to occurin vivoin the mucus of CF patients. Two CF-emergent bacterial species,Inquilinus limosusandDolosigranulum pigrum, and the major pathogenPseudomonas aeruginosawere studied in terms of biofilm development and antibiotic susceptibilities underin vitroatmospheres with different oxygen availabilities. All species were able to developin vitrobiofilms under different oxygen-available environments, withD. pigrumaccumulating high amounts of biomass and respiratory activities. When established, biofilms were of difficult eradication, with antibiotics losing their effectiveness in comparison with the corresponding planktonic populations. Surprisingly, biofilms of each emergent organism displayed multidrug resistance under aerobic environments, enduring even in low-oxygen atmospheres. This study suggests a potential prospect on the impact of nonconventional organismsI. limosusandD. pigrumon CF lung infections, demonstrating capacity to adapt to biofilm mode of life under restricted-oxygen atmospheres resembling CF airways, which may ultimately endanger the efficacy of currently used antibiotic regimens.

Oxygen-dependent changes in binding partners and post-translational modifications regulate the abundance and activity of HIF-1α/2α

2021 ◽  
Vol 14 (692) ◽  
pp. eabf6685
Author(s):  
Leonard A. Daly ◽  
Philip J. Brownridge ◽  
Michael Batie ◽  
Sonia Rocha ◽  
Violaine Sée ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Fine Tuning ◽  
Protein Protein Interactions ◽  
Low Oxygen ◽  
Hypoxic Response ◽  
Post Translational Modifications ◽  
Functional Roles ◽  
Binding Partners ◽  
Oxygen Conditions

Cellular adaptation to low-oxygen environments is mediated in part by the hypoxia-inducible factors (HIFs). Like other transcription factors, the stability and transcriptional activity of HIFs—and consequently, the hypoxic response—are regulated by post-translational modifications (PTMs) and changes in protein-protein interactions. Our current understanding of PTM-mediated regulation of HIFs is primarily based on in vitro protein fragment–based studies typically validated in fragment-expressing cells treated with hypoxia-mimicking compounds. Here, we used immunoprecipitation-based mass spectrometry to characterize the PTMs and binding partners for full-length HIF-1α and HIF-2α under normoxic (21% oxygen) and hypoxic (1% oxygen) conditions. Hypoxia substantially altered the complexity and composition of the HIFα protein interaction networks, particularly for HIF-2α, with the hypoxic networks of both isoforms being enriched for mitochondrial proteins. Moreover, both HIFα isoforms were heavily covalently modified. We identified ~40 PTM sites composed of 13 different types of modification on both HIFα isoforms, including multiple cysteine modifications and an unusual phosphocysteine. More than 80% of the PTMs identified were not previously known and about half exhibited oxygen dependency. We further characterized an evolutionarily conserved phosphorylation of Ser31 in HIF-1α as a regulator of its transcriptional function, and we propose functional roles for Thr406, Thr528, and Ser581 in HIF-2α. These data will help to delineate the different physiological roles of these closely related isoforms in fine-tuning the hypoxic response.

scholarly journals Microbial Consumption of Atmospheric Isoprene in a Temperate Forest Soil

1998 ◽  
Vol 64 (1) ◽  
pp. 172-177 ◽  
Author(s):  
C. C. Cleveland ◽  
J. B. Yavitt
Keyword(s):  
Atmospheric Chemistry ◽  
Temperate Forest ◽  
Temperature Response ◽  
Strong Relationship ◽  
Dry Weight ◽  
Low Oxygen ◽  
Oxygen Conditions ◽  
Consumption Rates ◽  
Microbial Consumption

ABSTRACT Isoprene (2-methyl-1,3 butadiene) is a low-molecular-weight hydrocarbon emitted in large quantities to the atmosphere by vegetation and plays a large role in regulating atmospheric chemistry. Until now, the atmosphere has been considered the only significant sink for isoprene. However, in this study we performed both in situ and in vitro experiments with soil from a temperate forest near Ithaca, N.Y., that indicate that the soil provides a sink for atmospheric isoprene and that the consumption of isoprene is carried out by microorganisms. Consumption occurred rapidly in field chambers (672.60 ± 30.12 to 2,718.36 ± 86.40 pmol gdw−1 day−1) (gdw is grams [dry weight] of soil; values are means ± standard deviations). Subsequent laboratory experiments confirmed that isoprene loss was due to biological processes: consumption was stopped by autoclaving the soil; consumption rates increased with repeated exposure to isoprene; and consumption showed a temperature response consistent with biological activity (with an optimum temperature of 30°C). Isoprene consumption was diminished under low oxygen conditions (120 ± 7.44 versus 528.36 ± 7.68 pmol gdw−1 day−1 under ambient O2concentrations) and showed a strong relationship with soil moisture. Isoprene-degrading microorganisms were isolated from the site, and abundance was calculated as 5.8 × 105 ± 3.2 × 105 cells gdw−1. Our results indicate that soil may provide a significant biological sink for atmospheric isoprene.

Postnatal weight of calves derived from in vitro matured and in vitro fertilized embryos developed under various oxygen concentrations

2000 ◽  
Vol 12 (8) ◽  
pp. 391 ◽  
Author(s):  
H. Iwata ◽  
N. Minami ◽  
H. Imai
Keyword(s):  
Oxygen Tension ◽  
Blastocyst Stage ◽  
High Oxygen ◽  
Gestation Length ◽  
Low Oxygen ◽  
Cell Stage ◽  
Fluid Medium ◽  
Significant Difference ◽  
Oxygen Conditions

In the present study, weights of calves (14 days after birth) derived from embryos of a homogeneous line (Tajima line) of Japanese Black Cow, cultured in vitro under various oxygen conditions was examined. In vitro matured and fertilized oocytes were incubated for 48 h in modified synthetic oviduct fluid medium under 5% CO 2in air and embryos reaching at least the 5-cell stage were selected for further culture under various gas conditions (high oxygen tension: 5% CO 2 in air; low oxygen tension: 5% O 2 , 5% CO 2 , 90% N 2 ) for 5 days. Embryos that developed to the blastocyst stage were transferred to Holstein cows or cryopreserved until transfer. When embryos were cultured under high oxygen tension and cryopreserved, the weights of male calves (at 14 days) were significantly heavier than in the other groups. However, there was no significant difference in gestation lengths of male calves. In female calves, no difference was observed in either the weight or gestation length of calves irrespective of oxygen tension during the culture period or embryo conditions (fresh or frozen). From the results of the present study, it is suggested that the oxygen concentration during culture and cryopreservation synergistically induced the production of overweight male calves without influencing gestation length.

Culture of Mobilized Human CD34+ Cells in Hypoxic Conditions Improves Lentiviral Transduction Efficiency in SCID-Repopulating Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3545-3545
Author(s):  
Andre Larochelle ◽  
Hezhi Gan ◽  
Joshua R. Clevenger ◽  
Cynthia E. Dunbar
Keyword(s):  
Cell Cycle ◽  
Transduction Efficiency ◽  
Low Oxygen ◽  
Lentiviral Transduction ◽  
Hypoxic Conditions ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Oxygen Conditions ◽  
Cells Cultured

Abstract Under normal physiological conditions, hematopoietic stem cells (HSC) are sequestered in a hypoxic microenvironment in the bone marrow (BM), suggesting that low oxygen levels may play a fundamental role in the maintenance of normal stem cell function and protect these cells from the damaging effects of reactive oxygen species (ROS). In vitro culture of human BM CD34+ cells under hypoxic conditions has been shown to result in expansion of SCID-repopulating cells (SRC) as compared to culture under normoxic conditions (JCI112 (1); 126, 2003). We investigated whether culture of human mobilized CD34+ cells under low oxygen conditions (5% O2) could improve lentiviral transduction efficiency in SRC compared with culture under atmospheric O2 conditions (21%). G-CSF mobilized CD34+ cells from 4 healthy volunteers were prestimulated for 48 hours in the presence of cytokines (SCF, Flt-3 ligand and thrombopoietin) and subsequently transduced in fibronectin coated plates for 24 hours with SIN-lentiviral vectors carrying the GFP gene under the control of an EF1α promoter. In 3 experiments, cells were used for in vitro assays, including ROS, phenotypic, cell cycle, clonogenic and apoptosis assays. In one experiment, cells were injected intravenously in the tail vein of sublethally irradiated NOD/SCID IL2rγ −/− mice after transduction. Intracellular ROS levels increased more significantly in human CD34+ cells cultured for 3 days in 21% O2 compared with cells cultured in 5% O2. When cultures were maintained more than 3 days, ROS levels were similar between the 2 conditions. The levels of expansion of CD34+ cells compared with baseline were similar in hypoxia (3.9-fold) and normoxia (3.5-fold) (p=0.47). In contrast, the expansion of CD34+CD38− cells, a subpopulation enriched in HSCs, was greater in hypoxia (3.8-fold) than in normoxia (2.2-fold) (p=0.02). After 3 days of culture, the total number of colony-forming cells (CFC) increased 1.1-fold and 1.3-fold under hypoxic and normoxic conditions, respectively (p=0.32) compared with freshly isolated CD34+ cells. The level of O2 had no significant effect on lineage commitment of the CFC. At baseline, the majority (59.5%) of the CD34+ cells were in the G0 phase of the cell cycle. After 3 days in culture under hypoxic or normoxic conditions, the percentages of cells in G0 were 5.5% and 3.5%, respectively (p=0.03). The differences in percentages of cells in the G1 and G2/S/M phases of the cell cycle were not statistically different. The percentages of CD34+ apoptotic cells were similar between hypoxic (32.8%) and normoxic (29.5%) conditions (p=0.18). The pO2 also had no impact on CD34+ cell death (12.2% at 5% O2 and 11.7% at 21% O2, p=0.9). When considering the bulk of CD34+ cells after transduction with GFP-lentiviral vectors, there was no statistically significant difference in the percentages of GFP+ cells under hypoxia (22.3%) or normoxia (21%) (p=0.88). In contrast, when CD34+ cells cultured under hypoxia were injected into NOD/SCID IL2rγ −/− mice at the end of the transduction period, improved human cell engraftment and lentiviral transduction efficiency were detected 2 months after transplantation compared with CD34+ cells cultured under normoxia. Human cell engraftment in the mouse BM, as determined by flow cytometry using a human specific CD45 antibody, was 84% in the hypoxic group (n=4) and 54% in the normoxic group (n=4) (p=0.04). The level of O2 had no significant impact on the lineage commitment of the SRC, with a majority of CD45+CD15+ granulocytes in both groups. The percentage of GFP+CD45+ cells was 54% (hypoxia) and 43% (normoxia) (p=0.02), indicating an improved transduction efficiency of SRC under hypoxic conditions. Overall, these data indicate that human CD34+ cells cultured under low oxygen conditions maintain a more primitive phenotype and have an increased susceptibility to lentiviral transduction compared with cells cultured in 21% O2 conditions. Improved engraftment and transduction efficiency do not appear to be related to decreased apoptosis in lower O2 concentrations; instead, increased ROS production in higher O2 concentrations could lead to increased cell signaling and differentiation. Use of low O2 levels for in vitro transduction of human CD34+ cells could have important clinical implications in gene therapy.

Towards Engineering Whole Bones via Endochondral Ossification

2013 ◽  
Author(s):  
Eamon J. Sheehy ◽  
Tatiana Vinardell ◽  
Conor T. Buckley ◽  
Daniel J. Kelly
Keyword(s):  
Tissue Engineering ◽  
Bone Tissue Engineering ◽  
Bone Tissue ◽  
Three Dimensional ◽  
Low Oxygen ◽  
Engineering Applications ◽  
Endochondral Bone ◽  
Oxygen Conditions

Tissue engineering applications aim to replace or regenerate damaged tissues through a combination of cells, three-dimensional scaffolds, and signaling molecules [1]. The endochondral approach to bone tissue engineering [2], which involves remodeling of an intermittent hypertrophic cartilaginous template, may be superior to the traditional intramembranous approach. Naturally derived hydrogels have been used extensively in tissue engineering applications [3]. Mesenchymal stem cell (MSC) seeded hydrogels may be a particularly powerful tool in scaling-up engineered endochondral bone grafts as the low oxygen conditions that develop within large constructs enhance in vitro chondrogenic differentiation and functional development [4]. A key requirement however, is that the hydrogel must allow for remodeling of the engineered hypertrophic cartilage into bone and also facilitate vascularization of the graft. The first objective of this study was to compare the capacity of different naturally derived hydrogels (alginate, chitosan, and fibrin) to generate in vivo endochondral bone. The secondary objective was to investigate the possibility of engineering a ‘scaled-up’ anatomically accurate distal phalange as a paradigm for whole bone tissue engineering.

scholarly journals Efficacy of Quinoxaline-2-Carboxylate 1,4-Di-N-Oxide Derivatives in Experimental Tuberculosis

2008 ◽  
Vol 52 (9) ◽  
pp. 3321-3326 ◽  
Author(s):  
Esther Vicente ◽  
Raquel Villar ◽  
Asunción Burguete ◽  
Beatriz Solano ◽  
Silvia Pérez-Silanes ◽  
...  
Keyword(s):  
Low Dose ◽  
Low Oxygen ◽  
Antitubercular Drugs ◽  
Aerosol Model ◽  
In Vivo Assays ◽  
Single Drug ◽  
Quinoxaline Derivatives ◽  
Oxygen Conditions

ABSTRACT This study extends earlier reports regarding the in vitro efficacies of the 1,4-di-N-oxide quinoxaline derivatives against Mycobacterium tuberculosis and has led to the discovery of a derivative with in vivo efficacy in the mouse model of tuberculosis. Quinoxaline-2-carboxylate 1,4-di-N-oxide derivatives were tested in vitro against a broad panel of single-drug-resistant M. tuberculosis strains. The susceptibilities of these strains to some compounds were comparable to those of strain H37Rv, as indicated by the ratios of MICs for resistant and nonresistant strains, supporting the premise that 1,4-di-N-oxide quinoxaline derivatives have a novel mode of action unrelated to those of the currently used antitubercular drugs. Specific derivatives were further evaluated in a series of in vivo assays, including evaluations of the maximum tolerated doses, the levels of oral bioavailability, and the efficacies in a low-dose aerosol model of tuberculosis in mice. One compound, ethyl 7-chloro-3-methylquinoxaline-2-carboxylate 1,4-dioxide, was found to be (i) active in reducing CFU counts in both the lungs and spleens of infected mice following oral administration, (ii) active against PA-824-resistant Mycobacterium bovis, indicating that the pathway of bioreduction/activation is different from that of PA-824 (a bioreduced nitroimidazole that is in clinical trials), and (iii) very active against nonreplicating bacteria adapted to low-oxygen conditions. These data indicate that 1,4-di-N-oxide quinoxalines hold promise for the treatment of tuberculosis.

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