SOGGY: solvent-optimized double gradient spectroscopy for water suppression. A comparison with some existing techniques.
ABSTRACT Excitation sculpting, a general method to suppress unwanted magnetization while controlling the phase of the retained signal [T.L. Hwang, A.J. Shaka, Water suppression that works. Excitation sculpting using arbitrary waveforms and pulsed field gradients, J. Magn. Reson. Ser. A 112 (1995) 275-279] is a highly effective method of water suppression for both biological and small molecule NMR spectroscopy. In excitation sculpting, a double pulsed field gradient spin echo forms the core of the sequence and pairing a low-power soft 180 degrees (-x) pulse with a high-power 180 degrees (x) all resonances except the water are flipped and retained, while the water peak is attenuated. By replacing the hard 180 degrees pulse in the double echo with a new phase-alternating composite pulse, broadband and adjustable excitation of large bandwidths with simultaneous high water suppression is obtained. This "Solvent-Optimized Gradient-Gradient Spectroscopy" (SOGGY) sequence is a reliable workhorse method for a wide range of practical situations in NMR spectroscopy, optimizing both solute sensitivity and water suppression.
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ABSTRACT: Aberrant energy metabolism is a hallmark of cancer. To fulfill the increased energy requirements, tumor cells secrete cytokines/factors inducing muscle and fat degradation in cancer patients, a condition known as cancer cachexia. It accounts for nearly 20% of all cancer-related deaths. However, the mechanistic basis of cancer cachexia and therapies targeting cancer cachexia thus far remain elusive. A ketogenic diet, a high-fat and low-carbohydrate diet that elevates circulating levels of ketone bodies (i.e., acetoacetate, β-hydroxybutyrate, and acetone), serves as an alternative energy source. It has also been proposed that a ketogenic diet leads to systemic metabolic changes. Keeping in view the significant role of metabolic alterations in cancer, we hypothesized that a ketogenic diet may diminish glycolytic flux in tumor cells to alleviate cachexia syndrome and, hence, may provide an efficient therapeutic strategy.Cancer & metabolism. 01/2014; 2:18.
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ABSTRACT: The tricarboxylic acid cycle (TCA cycle) is a central metabolic pathway that provides energy, reducing potential and biosynthetic intermediates. In Staphylococcus aureus, TCA cycle activity is controlled by several regulators (e.g., CcpA, CodY, and RpiRc) in response to the availability of sugars, amino acids, and environmental stress. Developing a bioinformatic search for additional carbon catabolite-responsive regulators in S. aureus, we identified a LysR-type regulator, catabolite control protein E (CcpE), with homology to the Bacillus subtilis CcpC regulator. Inactivation of ccpE in S. aureus strain Newman revealed that CcpE is a positive transcriptional effector of the first two enzymes of the TCA cycle, aconitase (citB) and to a lesser extent citrate synthase (citZ). Consistent with the transcriptional data, aconitase activity dramatically decreased in the ccpE mutant relative to the wild-type strain. The effect of ccpE inactivation on citB transcription and the lesser effect on citZ transcription were also reflected in electrophoretic mobility shift assays where CcpE bound to the citB promoter but not the citZ promoter. Metabolomic studies showed that inactivation of ccpE resulted in increased intracellular concentrations of acetate, citrate, lactate, and alanine, consistent with a redirection of carbon away from the TCA cycle. Taken together, our data suggest that CcpE is a major direct positive regulator of the TCA cycle gene citB.Journal of Biological Chemistry 11/2013; · 4.65 Impact Factor
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ABSTRACT: Transcription factors are key regulators in both normal and pathological cell processes. Affecting the activity of these proteins is a promising strategy for understanding gene regulation and developing effective therapeutics. Co(III) Schiff base complexes ([Co(acacen)(L)2 ](+) where L=labile axial ligands) have been shown to be potent inhibitors of a number of zinc metalloproteins including Cys2 His2 zinc finger transcription factors. Inhibition by [Co(acacen)(L)2 ](+) of the target protein is believed to occur through a dissociative exchange of the labile axial ligands for histidine (His) residues essential for function. Here, we report a series of spectroscopic investigations with model peptides of zinc fingers that elucidate the interaction between [Co(acacen)(L)2 ](+) complexes and zinc finger transcription factors. Observed changes in NMR chemical shifts and 2D (1) H-(1) H NOESY NMR spectra demonstrate the preference of [Co(acacen)(L)2 ](+) complexes to coordinate His residues over other amino acids. The conformation of [Co(acacen)(L)2 ](+) upon His coordination was characterized by (1) H NMR spectroscopy, near-UV CD, and electronic absorption. These studies reveal that the resulting His-coordinated [Co(acacen)(L)2 ](+) complex possesses an octahedral structure. The effects of [Co(acacen)(L)2 ](+) complexes on the zinc-finger structure were assessed by the degree of hydrogen bonding (probed by 2D NMR spectroscopy) and secondary-structure profiles measured by far-UV CD. These structural studies demonstrate the ability of [Co(acacen)(L)2 ](+) complexes to disrupt the ββα structure of zinc fingers, resulting in primarily random-coil conformations. A mechanism is described wherein [Co(acacen)(L)2 ](+) complexes inhibit zinc finger transcription factor activity through selectively coordinating His residues in the zinc finger by dissociative ligand exchange and disrupting the ββα structural motif required for gene regulation.Chemistry 11/2013; · 5.93 Impact Factor