NMR Tube (nmr + tube)

Distribution by Scientific Domains

Selected Abstracts

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger

FEBS JOURNAL, Issue 14 2002
Stephan J. A. Hesse
Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+ -alginate beads. The fungus maintained constant cytoplasmic pH (pHcyt) and vacuolar pH (pHvac) values of 7.6 and 6.2, respectively, when the extracellular pH (pHex) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a ,pH over the cytoplasmic membrane of 6.6,6.7 was reached (pHex 0.7,0.8). Maintenance of these large pH differences was possible without increased respiration compared to pHex 5.8. Perfusion in the presence of various hexoses and pentoses (pHex 5.8) revealed that the magnitude of ,pH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger ,pH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m -chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 m CCCP), transient (2 m CCCP) or permanent (10 m CCCP) collapse of the vacuolar membrane ,pH. Nonlethal levels of the metabolic inhibitor azide (N3,, 0.1 mm) caused a transient decrease in pHcyt that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pHex (1.8) and a high acid-secreting capacity, pHcyt and pHvac values were still well maintained (pH 7.5 and 6.4, respectively). [source]

Crystallization and preliminary X-ray diffraction studies of the tetramerization domain derived from the human potassium channel Kv1.3

Andreas Winklmeier
The tetramerization domain (T1 domain) derived from the voltage-dependent potassium channel Kv1.3 of Homo sapiens was recombinantly expressed in Escherichia coli and purified. The crystals were first grown in an NMR tube in 150,mM potassium phosphate pH 6.5 in the absence of additional precipitants. The crystals showed I4 symmetry characteristic of the naturally occurring tetrameric assembly of the single subunits. A complete native data set was collected to 1.2, resolution at 100,K using synchrotron radiation. [source]

Development of a small-scale bioreactor: Application to in vivo NMR measurement

Dorra Gmati
Abstract A perfused bioreactor allowing in vivo NMR measurement was developed and validated for Eschscholtzia californica cells. The bioreactor was made of a 10-mm NMR tube. NMR measurement of the signal-to-noise ratio was optimized using a sedimented compact bed of cells that were retained in the bioreactor by a supporting filter. Liquid medium flow through the cell bed was characterized from a mass balance on oxygen and a dispersive hydrodynamic model. Cell bed oxygen demand for 4 h perfusion required a minimal medium flow rate of 0.8 mL/min. Residence time distribution assays at 0.8,2.6 mL/min suggest that the cells are subjected to a uniform nutrient environment along the cell bed. Cell integrity was maintained for all culture conditions since the release of intracellular esterases was not significant even after 4 h of perfusion. In vivo NMR was performed for 31P NMR and the spectrum can be recorded after only 10 min of spectral accumulation (500 scans) with peaks identified as G-6P, F-6P, cytoplasmic Pi, vacuolar Pi, ATP, and ADP,, ATP, and ADP,, NADP and NDPG, NDPG and ATP,. Cell viability was shown to be maintained as 31P chemical shifts were constant with time for all the identified nuclei, thus suggesting constant intracellular pH. 2004 Wiley Periodicals, Inc. [source]

A noninvasive technique for the measurement of the energetic state of free-suspension mammalian cells

M. Ben-Tchavtchavadze
Abstract A perfusion small-scale bioreactor allowing on-line monitoring of the cell energetic state was developed for free-suspension mammalian cells. The bioreactor was designed to perform in vivo nuclear magnetic resonance (NMR) spectroscopy, which is a noninvasive and nondestructive method that permits the monitoring of intracellular nutrient concentrations, metabolic precursors and intermediates, as well as metabolites and energy shuttles, such as ATP, ADP, and NADPH. The bioreactor was made of a 10-mm NMR tube following a fluidized bed design. Perfusion flow rate allowing for adequate oxygen supply was found to be above 0.79 mL min,1 for high-density cell suspensions (108 cells). Chinese hamster ovary (CHO) cells were studied here as model system. Hydrodynamic studies using coloration/decoloration and residence time distribution measurements were realized to perfect bioreactor design as well as to determine operating conditions bestowing adequate homogeneous mixing and cell retention in the NMR reading zone. In vivo 31P NMR was performed and demonstrated the small-scale bioreactor platform ability to monitor the cell physiological behavior for 30-min experiments. 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source]

Influence of dT20 and [d(AT)10]2 on Cisplatin Hydrolysis Studied by Two-Dimensional [1H,15N] HMQC NMR Spectroscopy

Jo Vinje Dr.
Abstract The influence of the presence of DNA on the kinetics of cisplatin (cis -[PtCl2(NH3)2]) aquation (replacement of Cl, by H2O) and anation (replacement of H2O by Cl,) involved in the hydrolysis of cisplatin have been determined by two-dimensional [1H,15N] HMQC NMR spectroscopy. Single-stranded dT20 and double-stranded [d(AT)10]2 oligonucleotides were used as DNA models, avoiding guanines which are known to react rapidly with aquated cisplatin forms. Reactions starting from cis -[PtCl2(15NH3)2], or from a stoichiometric mixture of cis -[Pt(15NH3)2(H2O)2]2+ and Cl, (all 0.5,mM PtII; in ionic strength, adjusted to 0.095,M or 0.011,M with NaClO4, pH between 3.0 and 4.0) were followed in an NMR tube in both the absence and presence of 0.7,mM dT20 or [d(AT)10]2. In the presence of dT20, we observed a slight and ionic-strength-independent decrease (15,20,%) of the first aquation rate constant, and a more significant decrease of the second anation rate constant. The latter was more important at low ionic strength, and can be explained by efficient condensation of cis -[Pt(15NH3)2(H2O)2]2+ on the surface of single-stranded DNA, in a region depleted of chloride anions. At low ionic strength, we observed an additional set of [1H,15N] HMQC spectral signals indicative of an asymmetric species of PtN2O2 coordination, and we assigned them to phosphate-bound monoadducts of cis -[Pt(15NH3)2(H2O)2]2+. Double-stranded [d(AT)10]2 slowed down the first aquation step also by approximately 15,%; however, we could not determine the influence on the second hydrolysis step because of a significant background reaction with cis -[Pt(NH3)2(H2O)2]2+. [source]