scholarly journals Cellular reductase activity in uncultivatedThiomargarita spp. assayed using a redox-sensitive dye

2017 ◽  
Author(s):  
Jake V. Bailey ◽  
Beverly E. Flood ◽  
Elizabeth Ricci ◽  
Nathalie Delherbe
Keyword(s):  
Metabolic Activity ◽  
Time Course ◽  
Experimental Validation ◽  
Reductase Activity ◽  
Electron Donors ◽  
Metabolic Plasticity ◽  
Hypoxic Conditions ◽  
Large Size ◽  
Genomic Studies ◽  
Genomic Results

ABSTRACTThe largest known bacteria,Thiomargarita spp.,have yet to be isolated in pure culture, but their large size allows for individual cells to be followed in time course experiments, or to be individually sorted for ‘omics-based investigations. Here we report a novel application of a tetrazolium-based dye that measures the flux of reductase production from catabolic pathways to investigate the metabolic activity of individual cells ofThiomargaritaspp. When coupled to microscopy, staining of the cells with a tetrazolium-formazan dye allows for metabolic responses inThiomargaritaspp. to be to be tracked in the absence of observable cell division. Additionally, the metabolic activity ofThiomargaritaspp. cells can be differentiated from the metabolism of other microbes in specimens that contain adherent bacteria. The results of our redox-dye-based assay suggests thatThiomargaritais the most metabolically versatile under anoxic conditions where it appears to express cellular reductase activity in response to the electron donors succinate, acetate, citrate, formate, thiosulfate, H2, and H2S. Under hypoxic conditions, formazan staining results suggest the metabolism of succinate, and likely acetate, citrate, and H2S. Cells incubated under oxic conditions showed the weakest formazan staining response, and then only to H2S, citrate, and perhaps succinate. These results provide experimental validation of recent genomic studies ofCa.Thiomargarita nelsonii that suggest metabolic plasticity and mixotrophic metabolism. The cellular reductase response of bacteria attached to the exteriors ofThiomargaritaalso supports the possibility of trophic interactions between these largest of known bacteria and attached epibionts.IMPORTANCEThe metabolic potentials of many microorganisms that cannot be grown in the laboratory are known only from genomic data. Genomes ofThiomargaritaspp. suggest that these largest of known bacteria are mixotrophs, combining lithotrophic metabolisms with organic carbon degradation. Our use of a redox-sensitive tetrazolium dye to query the metabolism of these bacteria provides an independent line of evidence that corroborates the apparent metabolic plasticity ofThiomargaritaobserved in recently produced genomes. Finding new cultivation- independent means of testing genomic results is critical to testing genome-derived hypotheses on the metabolic potentials of uncultivated microorganisms.

scholarly journals Imaging of Cellular Oxidoreductase Activity Suggests Mixotrophic Metabolisms in Thiomargarita spp

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Jake V. Bailey ◽  
Beverly E. Flood ◽  
Elizabeth Ricci ◽  
Nathalie Delherbe
Keyword(s):  
Organic Carbon ◽  
Time Course ◽  
Genomic Data ◽  
Metabolic Potential ◽  
Oxidoreductase Activity ◽  
Metabolic Plasticity ◽  
Hypoxic Conditions ◽  
Genomic Studies ◽  
Genomic Results ◽  
Carbon Degradation

ABSTRACT The largest known bacteria, Thiomargarita spp., have yet to be isolated in pure culture, but their large size allows for individual cells to be monitored in time course experiments or to be individually sorted for omics-based investigations. Here we investigated the metabolism of individual cells of Thiomargarita spp. by using a novel application of a tetrazolium-based dye that measures oxidoreductase activity. When coupled with microscopy, staining of the cells with a tetrazolium-formazan dye allows metabolic responses in Thiomargarita spp. to be to be tracked in the absence of observable cell division. Additionally, the metabolic activity of Thiomargarita sp. cells can be differentiated from the metabolism of other microbes in specimens that contain adherent bacteria. The results of our redox dye-based assay suggest that Thiomargarita is the most metabolically versatile under anoxic conditions, where it appears to express cellular oxidoreductase activity in response to the electron donors succinate, acetate, citrate, formate, thiosulfate, H2, and H2S. Under hypoxic conditions, formazan staining results suggest the metabolism of succinate and likely acetate, citrate, and H2S. Cells incubated under oxic conditions showed the weakest formazan staining response, and then only to H2S, citrate, and perhaps succinate. These results provide experimental validation of recent genomic studies of Candidatus Thiomargarita nelsonii that suggest metabolic plasticity and mixotrophic metabolism. The cellular oxidoreductase response of bacteria attached to the exterior of Thiomargarita also supports the possibility of trophic interactions between these largest of known bacteria and attached epibionts. IMPORTANCE The metabolic potential of many microorganisms that cannot be grown in the laboratory is known only from genomic data. Genomes of Thiomargarita spp. suggest that these largest of known bacteria are mixotrophs, combining lithotrophic metabolism with organic carbon degradation. Our use of a redox-sensitive tetrazolium dye to query the metabolism of these bacteria provides an independent line of evidence that corroborates the apparent metabolic plasticity of Thiomargarita observed in recently produced genomes. Finding new cultivation-independent means of testing genomic results is critical to testing genome-derived hypotheses on the metabolic potentials of uncultivated microorganisms. IMPORTANCE The metabolic potential of many microorganisms that cannot be grown in the laboratory is known only from genomic data. Genomes of Thiomargarita spp. suggest that these largest of known bacteria are mixotrophs, combining lithotrophic metabolism with organic carbon degradation. Our use of a redox-sensitive tetrazolium dye to query the metabolism of these bacteria provides an independent line of evidence that corroborates the apparent metabolic plasticity of Thiomargarita observed in recently produced genomes. Finding new cultivation-independent means of testing genomic results is critical to testing genome-derived hypotheses on the metabolic potentials of uncultivated microorganisms.

Time Course of Tumor Metabolic Activity During Chemoradiotherapy of Esophageal Squamous Cell Carcinoma and Response to Treatment

2004 ◽  
Vol 22 (5) ◽  
pp. 900-908 ◽  
Author(s):  
Hinrich A. Wieder ◽  
Björn L.D.M. Brücher ◽  
Frank Zimmermann ◽  
Karen Becker ◽  
Florian Lordick ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Esophageal Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Metabolic Activity ◽  
Fdg Pet ◽  
Time Course ◽  
Tumor Response ◽  
Patient Survival ◽  
Resected Specimen

PurposeTo evaluate the time course of therapy-induced changes in tumor glucose use during chemoradiotherapy of esophageal squamous cell carcinoma (ESCC) and to correlate the reduction of metabolic activity with histopathologic tumor response and patient survival.Patients and MethodsThirty-eight patients with histologically proven intrathoracic ESCC (cT3, cN0/+, cM0) scheduled to undergo a 4-week course of preoperative simultaneous chemoradiotherapy followed by esophagectomy were included. Patients underwent positron emission tomography with the glucose analog fluorodeoxyglucose (FDG-PET) before therapy (n = 38), after 2 weeks of initiation of therapy (n = 27), and preoperatively (3 to 4 weeks after chemoradiotherapy; n = 38). Tumor metabolic activity was quantitatively assessed by standardized uptake values (SUVs).ResultsMean tumor FDG uptake before therapy was 9.3 ± 2.8 SUV and decreased to 5.7 ± 1.9 SUV 14 days after initiation of chemoradiotherapy (−38% ± 18%; P < .0001). The preoperative scan showed an additional decrease of metabolic activity to 3.3 ± 1.1 SUV (P < .0001). In histopathologic responders (< 10% viable cells in the resected specimen), the decrease in SUV from baseline to day 14 was 44% ± 15%, whereas it was only 21% ± 14% in nonresponders (P = .0055). Metabolic changes at this time point were also correlated with patient survival (P = .011). In the preoperative scan, tumor metabolic activity had decreased by 70% ± 11% in histopathologic responders and 51% ± 21% in histopathologic nonresponders.ConclusionChanges in tumor metabolic activity after 14 days of preoperative chemoradiotherapy are significantly correlated with tumor response and patient survival. This suggests that FDG-PET might be used to identify nonresponders early during neoadjuvant chemoradiotherapy, allowing for early modifications of the treatment protocol.

Persistence of mitochondrial competence during myocardial autolysis

1987 ◽  
Vol 252 (5) ◽  
pp. H985-H989
Author(s):  
W. Rouslin
Keyword(s):  
Membrane Potential ◽  
Time Course ◽  
Reductase Activity ◽  
Electron Flow ◽  
Coenzyme Q ◽  
Full Potential ◽  
Irreversible Loss ◽  
Control Activity ◽  
Inner Membrane ◽  
Potential Development

The rate of irreversible loss of mitochondrial phosphorylating respiratory function with NAD-linked substrates during zero flow myocardial autolysis at 37 degrees C was gradual and relatively linear with time, progressing at about 1% of the control activity per minute. State 3 respiratory rates and initial rates of inner membrane potential development dropped off in close parallel with one another as well as with NADH-coenzyme Q (CoQ) reductase activity, suggesting that oxygen uptake as well as membrane potential development were rate limited by the increasing impairment of electron flow through complex I. Although the initial rate of membrane potential development dropped off gradually, the time course for the loss of the ability to ultimately develop and hold a full potential was slower still, there being only a moderate impairment of this ability at 80 min of autolysis. This sustained ability to develop and hold a membrane potential after more than 1 h of autolysis suggested that inner membrane leakiness contributed little or not at all to the functional impairment observed. The irreversible loss of mitochondrial inner membrane competence emerged in these studies as a relatively late development in the sequence of cellular alterations which characterize the myocardial ischemic process.

An Improved RV Based Fuzzy Cluster Analysis (IRVFCA) Method for Task-Related Functional MRI Data Analysis

2013 ◽  
Vol 427-429 ◽  
pp. 1570-1573
Author(s):  
Han Wang ◽  
Wei Ming Zeng
Keyword(s):  
Time Course ◽  
Experimental Validation ◽  
Brain Volume ◽  
Pearson Correlation ◽  
Fuzzy Cluster ◽  
Fmri Data ◽  
Data Sets ◽  
Fuzzy Cluster Analysis ◽  
Fuzzy Clustering Method ◽  
The Brain

Functional magnetic resonance imaging (fMRI) has become one of the important tools of functional connectivity studies of the human brain. Fuzzy clustering method (FCM) is a common method for analysis of FMRI data. Traditional FCA methods measure the similarity between the BOLD time course of a centroid and the ones of all other voxels in the brain on the basis of Pearson correlation coefficient. This article puts forward a multi-voxel-based similarity measure, an improved RV (IRV) measure, which takes the hypothesis into account that the function homogeneous voxels of brain volume are spatially clustered within a local region. Experimental validation is presented through four visual fMRI data sets which shows that the IRVFCA method not only has improved the speed of FCA, but has comparatively raised the accuracy of the method.

scholarly journals A new method for assaying rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and its application in a study of the effect of dietary cholesterol on this effect of dietary cholesterol on this enzyme

1977 ◽  
Vol 164 (3) ◽  
pp. 501-508 ◽  
Author(s):  
Y A Baqir ◽  
R Booth
Keyword(s):  
Rat Liver ◽  
Cholesterol Ester ◽  
Time Course ◽  
Coenzyme A ◽  
Reductase Activity ◽  
Dietary Cholesterol ◽  
New Method ◽  
Coa Reductase ◽  
Microsomal Cholesterol ◽  
Coenzyme A Reductase

A new method suitable for measuring rat liver 3-hydroxy-3-methylglutaryl-CoA reductase activity is described and its advantages over methods previously available are discussed. An accurate time course was measured for the inhibition of liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase activity by dietary cholesterol; this enzyme was affected 1 1/4 h after the rats began to consume a cholesterol-rich diet. In this experiment there was no correlation between concentrations of microsomal cholesterol ester and the activity of 3-hydroxy-3-methylglutary-CoA reductase.

In vitro and in vivo kinetic handling of nitrite in blood: effects of varying hemoglobin oxygen saturation

2007 ◽  
Vol 293 (3) ◽  
pp. H1508-H1517 ◽  
Author(s):  
Arlin B. Blood ◽  
Gordon G. Power
Keyword(s):  
Nitric Oxide ◽  
Time Course ◽  
Kinetic Properties ◽  
Hemoglobin Oxygen Saturation ◽  
Hypoxic Conditions ◽  
Oxygenation Condition ◽  
Plasma Nitrite ◽  
Kinetics Of

Growing evidence suggests that nitrite, acting via reduction to nitric oxide by deoxyhemoglobin, may play an important role in local control of blood flow during hypoxia. To investigate the effect of hypoxia (65 Torr arterial Po2) on the kinetic properties of nitrite, a bolus injection of sodium nitrite (10 mg/kg iv) was given to normoxic or hypoxic newborn lambs, and the time course of plasma nitrite and methemoglobin (MetHb) concentrations was measured. The in vivo kinetics of nitrite disappearance from plasma were biphasic and were not affected by hypoxia. Changes in MetHb, a product of the nitrite-hemoglobin reaction, also did not differ with the level of oxygenation. Hypoxia potentiated the hypotensive effects of nitrite on pulmonary and systemic arterial pressures. The disappearance of nitrite from plasma was equivalent to the increase in MetHb on a molar basis. In contrast, nitrite metabolism in sheep blood in vitro resulted in more than one MetHb per nitrite equivalent under mid- and high-oxygenation conditions: oxyhemoglobin (HbO2) saturation = 50.3 ± 1.7% and 97.0 ± 1.3%, respectively. Under the low-oxygenation condition (HbO2 saturation = 5.2 ± 0.9%), significantly less than 1 mol of MetHb was produced per nitrite equivalent, indicating that a significant portion of nitrite is metabolized through pathways that do not produce MetHb. These data support the idea that the vasodilating effects of nitrite are potentiated under hypoxic conditions due to the reduction of nitrite to nitric oxide by deoxyhemoglobin.

scholarly journals Spectroscopic analysis of myoglobin and cytochrome c dynamics in isolated cardiomyocytes during hypoxia and reoxygenation

2015 ◽  
Vol 12 (105) ◽  
pp. 20141339 ◽  
Author(s):  
A. Almohammedi ◽  
S. M. Kapetanaki ◽  
B. R. Wood ◽  
E. L. Raven ◽  
N. M. Storey ◽  
...  
Keyword(s):  
Dissolved Oxygen ◽  
Cellular Response ◽  
Ex Vivo ◽  
Reductase Activity ◽  
Sodium Dithionite ◽  
Raman Microspectroscopy ◽  
Chemical Agent ◽  
Rat Cardiomyocytes ◽  
Hypoxic Conditions ◽  
In Cells

Raman microspectroscopy was applied to monitor the intracellular redox state of myoglobin and cytochrome c from isolated adult rat cardiomyocytes during hypoxia and reoxygenation. The nitrite reductase activity of myoglobin leads to the production of nitric oxide in cells under hypoxic conditions, which is linked to the inhibition of mitochondrial respiration. In this work, the subsequent reoxygenation of cells after hypoxia is shown to lead to increased levels of oxygen-bound myoglobin relative to the initial levels observed under normoxic conditions. Increased levels of reduced cytochrome c in ex vivo cells are also observed during hypoxia and reoxygenation by Raman microspectroscopy. The cellular response to reoxygenation differed dramatically depending on the method used in the preceding step to create hypoxic conditions in the cell suspension, where a chemical agent, sodium dithionite, leads to reduction of cytochromes in addition to removal of dissolved oxygen, and bubbling-N 2 gas leads to displacement of dissolved oxygen only. These results have an impact on the assessment of experimental simulations of hypoxia in cells. The spectroscopic technique employed in this work will be used in the future as an analytical method to monitor the effects of varying levels of oxygen and nutrients supplied to cardiomyocytes during either the preconditioning of cells or the reperfusion of ischaemic tissue.

scholarly journals The Effect and Mechanism of Celecoxib in Hypoxia-Induced Survivin Up-Regulation in HUVECs

2015 ◽  
Vol 37 (3) ◽  
pp. 991-1001 ◽  
Author(s):  
Ning-ning Liu ◽  
Ning Zhao ◽  
Na Cai
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Time Course ◽  
Umbilical Vein ◽  
Survivin Expression ◽  
Mrna Levels ◽  
Cytoplasmic Staining ◽  
Hypoxic Conditions ◽  
Cox 2

Background/Aims: To investigate the roles of hypoxia-inducible factor 1α (HIF-1α), cyclooxygenase-2 (Cox-2) and its product, Prostaglandin E2 (PGE2), in the mechanisms underlying hypoxia-induced survivin expression in human umbilical vein endothelial cells (HUVECs) and to examine the effect of celecoxib, a selective Cox-2 inhibitor, on survivin expression. Methods: HUVECs were exposed to a normal (95% O2) or hypoxic (3% O2) environment for 24 hrs. We observed the localized expression of survivin, Cox-2 and HIF-1α in HUVECs using immunocytochemistry and detected the inhibitory effects of celecoxib on the growth of HUVECs using an MTT assay. mRNA and protein levels of Cox-2, HIF-1α and survivin were determined by real-time PCR and Western blot analysis under hypoxic conditions for 0, 6, 12, or 24 hrs. The time course changes of HIF-1α and survivin protein expression induced by cobalt chloride (CoCl2) were studied using Western blot analysis. We then treated HUVECs under hypoxia for 24 hrs with celecoxib (a Cox-2 selective inhibitor), genistein (a HIF-1α inhibitor) or exogenous PGE2 to further investigate the changes in hypoxia-induced survivin expression. Results: Following 24 hrs of hypoxic treatment, cells exhibited strongly positive survivin, HIF-1α and Cox-2 cytoplasmic staining. Celecoxib (65 μM) effectively inhibited cell proliferation under hypoxic conditions. The protein and mRNA levels of Cox-2, HIF-1α and survivin were increased under hypoxia. The patterns of HIF-1α and survivin expression induced by CoCl2 were similar to those induced by exposure to hypoxia. Genistein partially blocked survivin expression. Celecoxib reversed the hypoxia-induced survivin expression, whereas the addition of PGE2 partially restored this effect. Conclusions: Hypoxia-induced survivin expression in HUVECs may be mediated by dual interdependent mechanisms directly involving HIF-1α and indirectly involving the Cox-2/PGE2 pathways. Celecoxib may offset hypoxia-induced survivin expression.

scholarly journals Role of narK2X and narGHJI inHypoxic Upregulation of Nitrate Reduction byMycobacteriumtuberculosis

2003 ◽  
Vol 185 (24) ◽  
pp. 7247-7256 ◽  
Author(s):  
Charles D. Sohaskey ◽  
Lawrence G. Wayne
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Anaerobic Growth ◽  
Reporter System ◽  
Whole Cells ◽  
Hypoxic Conditions ◽  
Cell Extracts ◽  
Insertional Inactivation ◽  
Nitrate And Nitrite

ABSTRACT Mycobacterium tuberculosis is one of the strongest reducers of nitrate in the genus Mycobacterium. Under microaerobic conditions, whole cells exhibit upregulation of activity, producing approximately eightfold more nitrite than those of aerobic cultures of the same age. Assays of cell extracts from aerobic cultures and hypoxic cultures yielded comparable nitrate reductase activities. Mycobacterium bovis produced only low levels of nitrite, and this activity was not induced by hypoxia. M. tuberculosis has two sets of genes, narGHJI and narX of the narK2X operon, that exhibit some degree of homology to prokaryotic dissimilatory nitrate reductases. Each of these were knocked out by insertional inactivation. The narG mutant showed no nitrate reductase activity in whole culture or in cell-free assays, while the narX mutant showed wild-type levels in both assays. A knockout of the putative nitrite transporter narK2 gene produced a strain that had aerobic levels of nitrate reductase activity but failed to show hypoxic upregulation. Insertion of the M. tuberculosis narGHJI into a nitrate reductase Escherichia coli mutant allowed anaerobic growth in the presence of nitrate. Under aerobic and hypoxic conditions, transcription of narGHJI was constitutive, while the narK2X operon was induced under hypoxia, as measured with a lacZ reporter system and by quantitative real-time reverse PCR. This indicates that nitrate reductase activity in M. tuberculosis is due to the narGHJI locus with no detectable contribution from narX and that the hypoxic upregulation of activity is associated with the induction of the nitrate and nitrite transport gene narK2.

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