The use of metronidazole to study pyridine nucleotide reduction in vivo in Azotobacter vinelandii

1989 ◽  
Vol 35 (2) ◽  
pp. 340-343
Author(s):  
Jay B. Peterson
Keyword(s):  
Fluorescence Spectroscopy ◽  
Electron Acceptor ◽  
Azotobacter Vinelandii ◽  
Pyridine Nucleotide ◽  
Nitrogen Fixing ◽  
Whole Cell

Flavins of the particulate and supernatant fractions from nitrogen-fixing Azotobacter vinelandii cells were examined by fluorescence spectroscopy for reduction by NAD(P)H in the absence and presence of the low potential electron acceptor metronidazole. The objective was to determine if reduction of metronidazole-oxidized flavin in vivo results from NAD(P)H generation. Flavin was almost completely reduced by NADH, but NADPH gave less reduction. Most of the flavin was reduced in the presence of metronidazole. Menadione bisulfite prevented reduction. In whole-cell studies, mannitol-generated NAD(P)H reduced flavin in the presence of menadione bisulfite. These results establish that reduction of metronidazole-oxidized cell flavin in vivo is an indicator of NAD(P)H production.Key words: metronidazole, NAD(P)H reduction, Azotobacter vinelandii.

scholarly journals In Vivo Assembly of Artificial Metalloenzymes and Application in Whole‐Cell Biocatalysis**

2021 ◽  
Author(s):  
Shreyans Chordia ◽  
Siddarth Narasimhan ◽  
Alessandra Lucini Paioni ◽  
Marc Baldus ◽  
Gerard Roelfes
Keyword(s):  
Whole Cell ◽  
Whole Cell Biocatalysis ◽  
Artificial Metalloenzymes

scholarly journals In VitroandIn VivoCharacterization of the Novel Oxabicyclooctane-Linked Bacterial Topoisomerase Inhibitor AM-8722, a Selective, Potent Inhibitor of Bacterial DNA Gyrase

2016 ◽  
Vol 60 (8) ◽  
pp. 4830-4839 ◽  
Author(s):  
Christopher M. Tan ◽  
Charles J. Gill ◽  
Jin Wu ◽  
Nathalie Toussaint ◽  
Jingjun Yin ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Antibacterial Agents ◽  
Cell Activity ◽  
Dna Gyrase ◽  
Topoisomerase Iv ◽  
Bacterial Dna ◽  
Whole Cell ◽  
Content Type

ABSTRACTOxabicyclooctane-linked novel bacterial topoisomerase inhibitors (NBTIs) represent a new class of recently described antibacterial agents with broad-spectrum activity. NBTIs dually inhibit the clinically validated bacterial targets DNA gyrase and topoisomerase IV and have been shown to bind distinctly from known classes of antibacterial agents directed against these targets. Herein we report the molecular, cellular, andin vivocharacterization of AM-8722 as a representative N-alkylated-1,5-naphthyridone left-hand-side-substituted NBTI. Consistent with its mode of action, macromolecular labeling studies revealed a specific effect of AM-8722 to dose dependently inhibit bacterial DNA synthesis. AM-8722 displayed greater intrinsic enzymatic potency than levofloxacin versus both DNA gyrase and topoisomerase IV fromStaphylococcus aureusandEscherichia coliand displayed selectivity against human topoisomerase II. AM-8722 was rapidly bactericidal and exhibited whole-cell activity versus a range of Gram-negative and Gram-positive organisms, with no whole-cell potency shift due to the presence of DNA or human serum. Frequency-of-resistance studies demonstrated an acceptable rate of resistance emergencein vitroat concentrations 16- to 32-fold the MIC. AM-8722 displayed acceptable pharmacokinetic properties and was shown to be efficacious in mouse models of bacterial septicemia. Overall, AM-8722 is a selective and potent NBTI that displays broad-spectrum antimicrobial activityin vitroandin vivo.

scholarly journals ADR1-Mediated Transcriptional Activation Requires the Presence of an Intact TFIID Complex

1998 ◽  
Vol 18 (10) ◽  
pp. 5861-5867 ◽  
Author(s):  
Philip B. Komarnitsky ◽  
Edward R. Klebanow ◽  
P. Anthony Weil ◽  
Clyde L. Denis
Keyword(s):  
Transcriptional Activation ◽  
Yeast Cells ◽  
Binding Studies ◽  
Whole Cell ◽  
Transactivation Domains ◽  
Cell Extracts ◽  
Genes Expression ◽  
Tfiid Complex

ABSTRACT The yeast transcriptional activator ADR1, which is required forADH2 and other genes’ expression, contains four transactivation domains (TADs). While previous studies have shown that these TADs act through GCN5 and ADA2, and presumably TFIIB, other factors are likely to be involved in ADR1 function. In this study, we addressed the question of whether TFIID is also required for ADR1 action. In vitro binding studies indicated that TADI of ADR1 was able to retain TAFII90 from yeast extracts and TADII could retain TBP and TAFII130/145. TADIV, however, was capable of retaining multiple TAFIIs, suggesting that TADIV was binding TFIID from yeast whole-cell extracts. The ability of TADIV truncation derivatives to interact with TFIID correlated with their transcription activation potential in vivo. In addition, the ability of LexA-ADR1-TADIV to activate transcription in vivo was compromised by a mutation in TAFII130/145. ADR1 was found to associate in vivo with TFIID in that immunoprecipitation of either TAFII90 or TBP from yeast whole-cell extracts specifically coimmunoprecipitated ADR1. Most importantly, depletion of TAFII90 from yeast cells dramatically reducedADH2 derepression. These results indicate that ADR1 physically associates with TFIID and that its ability to activate transcription requires an intact TFIID complex.

Optimal conditions for induction of competence in nitrogen-fixing Azotobacter vinelandii

1982 ◽  
Vol 28 (4) ◽  
pp. 389-397 ◽  
Author(s):  
William J. Page
Keyword(s):  
Dissolved Oxygen ◽  
Sodium Acetate ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Cell Mass ◽  
Apparent Competition ◽  
Competence Development ◽  
Optimal Conditions ◽  
Nitrogen Fixing ◽  
Ph Range

Competence development in nitrogen-fixing Azotobacter vinelandii cells was optimal at pH 7.2–7.4 which necessitated additional buffering of the iron-limited nitrogen-free competence medium or the addition of a suitable organic acid salt, e.g., sodium acetate. An autolysin was active in this pH range and competent cells were more susceptible to autolysis than the general cell population. Competence development also required restricted aeration of the culture, and only those cultures that attained zero dissolved oxygen became competent. Restricted aeration served to protect the iron-limited cell nitrogenase from oxygen inactivation thus allowing the culture to reach zero dissolved oxygen. The inclusion of additional sources of reductant, e.g., malate, in buffered competence medium resulted in increased respiration and protection of nitrogenase, increased cell mass, and poly-β-hydroxybutyrate synthesis, but decreased competence. A possible explanation for the apparent competition between competence development and nitrogenase activity is discussed.

scholarly journals Compensation of physiological motion enables high-yield whole-cell recording in vivo

2020 ◽  
Author(s):  
William M. Stoy ◽  
Bo Yang ◽  
Ali Kight ◽  
Nathaniel C. Wright ◽  
Peter Y. Borden ◽  
...  
Keyword(s):  
Single Cells ◽  
Strong Dependence ◽  
High Yield ◽  
Patch Clamp Recording ◽  
Gold Standard Method ◽  
Whole Cell ◽  
Whole Cell Recording ◽  
Cell Recording ◽  
Living Mouse

1.1.1AbstractWhole-cell patch-clamp recording in vivo is the gold-standard method for measuring subthreshold electrophysiology from single cells during behavioural tasks, sensory stimulations, and optogenetic manipulation. However, these recordings require a tight, gigaohm resistance, seal between a glass pipette electrode’s aperture and a cell’s membrane. These seals are difficult to form, especially in vivo, in part because of a strong dependence on the distance between the pipette aperture and cell membrane. We elucidate and utilize this dependency to develop an autonomous method for placement and synchronization of pipette’s tip aperture to the membrane of a nearby, moving neuron, which enables high-yield seal formation and subsequent recordings in the deep in the brain of the living mouse, in the thalamus. This synchronization procedure nearly doubles the reported gigaseal yield in the thalamus (>3 mm below the pial surface) from 26% (n=17/64) to 48% (n=32/66). Whole-cell recording yield improved from 10% (n = 9/88) to 24% (n=18/76) when motion compensation was used during the gigaseal formation. As an example of its application, we utilized this system to investigate the role of the sensory environment and ventral posterior medial region (VPM) projection synchrony on intracellular dynamics in the barrel cortex. This method results in substantially greater subcortical whole-cell recording yield than previously reported and thus makes pan-brain whole-cell electrophysiology practical in the living mouse brain.

The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia eutropha

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2301-2311 ◽  
Author(s):  
Markus Pötter ◽  
Helena Müller ◽  
Frank Reinecke ◽  
Roman Wieczorek ◽  
Florian Fricke ◽  
...  
Keyword(s):  
Ralstonia Eutropha ◽  
Azotobacter Vinelandii ◽  
Production Systems ◽  
Complex Structure ◽  
Pcr Analysis ◽  
Unknown Protein ◽  
Considerable Impact ◽  
Unexpected Findings

Analysis of the genome sequence of the polyhydroxyalkanoate- (PHA) accumulating bacterium Ralstonia eutropha strain H16 revealed three homologues (PhaP2, PhaP3 and PhaP4) of the phasin protein PhaP1. PhaP1 is known to constitute the major component of the layer at the surface of poly(3-hydroxybutyrate), poly(3HB), granules. PhaP2, PhaP3 and PhaP4 exhibited 42, 49 and 45 % identity or 61, 62 and 63 % similarity to PhaP1, respectively. The calculated molecular masses of PhaP1, PhaP2, PhaP3 and PhaP4 were 20·0, 20·2, 19·6 and 20·2 kDa, respectively. RT-PCR analysis showed that phaP2, phaP3 and phaP4 were transcribed under conditions permissive for accumulation of poly(3HB). 2D PAGE of the poly(3HB) granule proteome and analysis of the detected proteins by MALDI-TOF clearly demonstrated that PhaP1, PhaP3 and PhaP4 are bound to the poly(3HB) granules in the cells. PhaP3 was expressed at a significantly higher level in PhaP1-negative mutants. Occurrence of an unknown protein with an N-terminal amino-acid sequence identical to that of PhaP2 in crude cellular extracts of R. eutropha had previously been shown by others. Although PhaP2 could not be localized in vivo on poly(3HB) granules, in vitro experiments clearly demonstrated binding of PhaP2 to these granules. Further analysis of complete or partial genomes of other poly(3HB)-accumulating bacteria revealed the existence of multiple phasin homologues in Ralstonia solanacearum, Burkholderia fungorum and Azotobacter vinelandii. These new and unexpected findings should affect our current models of PHA-granule structure and may also have a considerable impact on the establishment of heterologous production systems for PHAs.

Sign in / Sign up

Export Citation Format

Share Document