Citations

The magnetization exchange between two groups of equivalent spins each having different populations and overall relaxation rates has been analyzed. The results suggest that either the spin population difference or the overall relaxation rate difference in an exchange spectrum can produce cross-peaks with volumes larger than that of the corresponding diagonal line. This is important for interpretation of the magnetization exchange between water and macromolecular protons where both the population differences and auto-relaxation rate differences can be very large. Theoretical predictions of peak volume evolution were experimentally verified in the intermolecular magnetization exchange between the water and labile amide proton in a model system N-acetylglycine/water.

ABSTRACT Metabolic injury is a complex process affecting various tissues, with intracellular Ca 2+ loading recognized as a common precipitating event leading to cell death. We have recently observed that cells overexpressing recombinant ATP‐sensitive K + (K ATP ) channel subunits may acquire resistance against metabolic stress. To examine whether, under metabolic challenge, intracellular Ca 2+ homeostasis can be maintained by an activator of channel proteins, we delivered Kir6.2 and SUR2A genes, which encode K ATP channel subunits, into a somatic cell line lacking native K ATP channels. Hypoxia‐reoxygenation was simulated by application and removal of the mitochondrial poison 2,4 dinitrophenol. Under such metabolic stress, Ca 2+ loading was induced by Ca 2+ influx during hypoxia and release of Ca 2+ from intracellular stores during reoxygenation. Delivery of Kir6.2/SUR2A genes, in conjunction with the K ATP channel activator pinacidil, prevented intracellular Ca 2+ loading irrespective of whether the channel opener was applied throughout the duration of hypoxia‐reoxygenation or transiently during the hypoxic or reoxygenation stage. In all stages of injury, the effect of pinacidil was inhibited by the selective antagonist of K ATP channel, 5‐hydroxydecanoate. The present study provides evidence that combined use of gene delivery and pharmacological targeting of recombinant proteins can handle intracellular Ca 2+ homeostasis under hypoxia‐reoxygenation irrespective of the stage of the metabolic insult.—Jovanović, N., Jovanović, S., Jovanović, A., Terzic, A. Gene delivery of Kir6.2/SUR2A in conjunction with pinacidil handles intracellular Ca 2+ homeostasis under metabolic stress. FASEB J. 13, 923–929 (1999)

Brain mutations, young and old Most neurons that make up the human brain are postmitotic, living and functioning for a very long time without renewal (see the Perspective by Lee). Bae et al. examined the genomes of single neurons from the prenatal developing human brain. Both the type of mutation and the rates of accumulation changed between gastrulation and neurogenesis. These early mutations could be generating useful neuronal diversity or could predispose individuals to later dysfunction. Lodato et al. also found that neurons take on somatic mutations as they age by sequencing single neurons from subjects aged 4 months to 82 years. Somatic mutations accumulated with increasing age and accumulated faster in individuals affected by inborn errors in DNA repair. Postmitotic mutations might only affect one neuron, but the accumulated divergence of genomes across the brain could affect function. Science , this issue p. 550 , p. 555 ; see also p. 521

Single-cell diversity in the brain The cells that make up an organism may all start from one genome, but somatic mutations mean that somewhere along the line of development, an organism's individual cellular genomes diverge. McConnell et al. review the implications and causes of single-cell genomic diversity for brain function. Somatic mutations caused by mobile genetic elements or errors in DNA repair may underlie certain neuropsychiatric disorders. Science , this issue p. eaal1641

Deletion of the major adenylate kinase AK1 isoform, which catalyzes adenine nucleotide exchange, disrupts cellular energetic economy and compromises metabolic signal transduction. However, the consequences of deleting the AK1 gene on cardiac energetic dynamics and performance in the setting of ischemia-reperfusion have not been determined. Here, at the onset of ischemia, AK1 knockout mice hearts displayed accelerated loss of contractile force compared with wild-type controls, indicating reduced tolerance to ischemic stress. On reperfusion, AK1 knockout hearts demonstrated reduced nucleotide salvage, resulting in lower ATP, GTP, ADP, and GDP levels and an altered metabolic steady state associated with diminished ATP-to-Piand creatine phosphate-to-Piratios. Postischemic AK1 knockout hearts maintained ∼40% of β-phosphoryl turnover, suggesting increased phosphotransfer flux through remaining adenylate kinase isoforms. This was associated with sustained creatine kinase flux and elevated cellular glucose-6-phosphate levels as the cellular energetic system adapted to deletion of AK1. Such metabolic rearrangements, along with sustained ATP-to-ADP ratio and total ATP turnover rate, maintained postischemic contractile recovery of AK1 knockout hearts at wild-type levels. Thus deletion of the AK1 gene reveals that adenylate kinase phosphotransfer supports myocardial function on initiation of ischemic stress and safeguards intracellular nucleotide pools in postischemic recovery.

Next-generation screening of disease-related metabolomic phenotypes requires monitoring of both metabolite levels and turnover rates. Stable isotope18O-assisted31P nuclear magnetic resonance (NMR) and mass spectrometry uniquely allows simultaneous measurement of phosphometabolite levels and turnover rates in tissue and blood samples. The18O labeling procedure is based on the incorporation of one18O into Pifrom [18O]H2O with each act of ATP hydrolysis and the distribution of18O-labeled phosphoryls among phosphate-carrying molecules. This enables simultaneous recording of ATP synthesis and utilization, phosphotransfer fluxes through adenylate kinase, creatine kinase, and glycolytic pathways, as well as mitochondrial substrate shuttle, urea and Krebs cycle activity, glycogen turnover, and intracellular energetic communication. Application of expanded18O-labeling procedures has revealed significant differences in the dynamics of G-6-P[18O] (glycolysis), G-3-P[18O] (substrate shuttle), and G-1-P[18O] (glycogenolysis) between human and rat atrial myocardium. In human atria, the turnover of G-3-P[18O], which defects are associated with the sudden death syndrome, was significantly higher indicating a greater importance of substrate shuttling to mitochondria. Phosphometabolomic profiling of transgenic hearts deficient in adenylate kinase (AK1−/−), which altered levels and mutations are associated to human diseases, revealed a stress-induced shift in metabolomic profile with increased CrP[18O] and decreased G-1-P[18O] metabolic dynamics. The metabolomic profile of creatine kinase M-CK/ScCKmit−/−-deficient hearts is characterized by a higher G-6-[18O]P turnover rate, G-6-P levels, glycolytic capacity, γ/β-phosphoryl of GTP[18O] turnover, as well as β-[18O]ATP and β-[18O]ADP turnover, indicating altered glycolytic, guanine nucleotide, and adenylate kinase metabolic flux. Thus,18O-assisted gas chromatography-mass spectrometry and31P NMR provide a suitable platform for dynamic phosphometabolomic profiling of the cellular energetic system enabling prediction and diagnosis of metabolic diseases states.

In this study, the first complexes of Zn(II) with the N-benzyloxycar- bonyl-S-alaninato ligand (N-Boc-S-ala) were synthesized. The new complexes were characterized by elemental analysis, conductometric measurements, IR, 1H-NMR, 13C-NMR and 2D-NMR spectroscopy. On the basis of the experimental data, tetrahedral geometry of the Zn(II) complexes was proposed. A very good agreement between the NMR and DFT calculated data was obtained. Investigation of antimicrobial activity of the newly synthesized complexes was also performed. It was established that [Zn(N-Boc-S-ala)2] was selective and acts only on Candida albicans.