(1)H-NMR spectroscopy at 500 MHz was used to confirm that a previously unidentified singlet resonance at 3.14 ppm in the spectra of cerebrospinal fluid and plasma samples corresponds to dimethyl sulfone (DMSO(2)). A triple resonance inverse cryogenic NMR probe, with pre-amplifier and the RF-coils cooled to low temperature, was used to obtain an (1)H-(13)C HSQC spectrum of CSF containing 8 microM (753 ng/ml) DMSO(2). The (1)H-(13)C correlation signal for DMSO(2) was assigned by comparison with the spectrum from an authentic reference sample. In plasma and CSF from healthy controls, the concentration of DMSO(2) ranged between 0 and 25 micromol/l. The concentration of DMSO(2) in plasma from three of four patients with severe methionine adenosyltransferase I/III (MAT I/III) deficiency was about twice the maximum observed for controls. Thus, DMSO(2) occurs as a regular metabolite at low micromolar concentrations in cerebrospinal fluid and plasma. It derives from dietary sources, from intestinal bacterial metabolism and from human endogenous methanethiol metabolism.

A diagnosis of 3-methylglutaconic aciduria type I (OMIM: 250950) based on elevated urinary excretion of 3-methylglutaconic acid (3MGA), 3-methylglutaric acid (3MG) and 3-hydroxyisovaleric acid (3HIVA) was made in a 61-year-old female patient presenting with leukoencephalopathy slowly progressing over more than 30 years. The diagnosis was confirmed at the enzymatic and molecular level. In vivo brain MR spectroscopic imaging (MRSI) was performed at 3.0 T, and one-dimensional and two-dimensional in vitro NMR spectroscopy of body fluids of the patient was performed at 11.7 T. Additionally, we measured 1D (1)H-NMR spectra of urine of seven patients with a total of four different inborn errors of leucine metabolism. Increased concentrations of 3HIVA, 3MGA (cis and trans) and 3MG were observed in the NMR spectra of the patient's urine. In the cerebrospinal fluid, the 3HIVA concentration was 10 times higher than in the plasma of the patient and only the cis isomer of 3MGA was observed. In vivo brain MRSI showed an abnormal resonance at 1.28 ppm that may be caused by 3HIVA. Comparison of (1)H-NMR spectra of urine samples from all eight patients studied, representing five different inborn errors of leucine metabolism, showed that each disease has typical NMR characteristics. Our leukoencephalopathy patient suffers from a late-onset form of 3-methylglutaconic aciduria type I. In the literature, only very few adult patients with this conditions have been described, and 3HIVA accumulation in white matter in the brain has not been presented before in these patients. Our data demonstrate that (1)H-NMR spectroscopy of urine can easily discriminate between the known inborn errors of leucine metabolism and provide the correct diagnosis.

No abstract

In prokaryotic transcription regulation, several host factors form a complex with RNA polymerase and the nascent mRNA. As part of a process known as antitermination, two of these host factors, NusB and NusE, bind to form a heterodimer, which interacts with a specific boxA site on the RNA. The NusB/NusE/boxA RNA ternary complex interacts with the RNA polymerase transcription complex, stabilizing it and allowing transcription past premature termination points. The NusB protein also binds boxA RNA individually and retains all specificity for boxA. However, NusE increases the affinity of RNA to NusB in the ternary complex, which contributes to efficient antitermination. To understand the molecular mechanism of the process, we have determined the structure of NusB from the thermophilic bacterium Aquifex aeolicus and studied the interaction of NusB and NusE. We characterize this binding interaction using NMR, isothermal titration calorimetry, gel filtration, and analytical ultracentrifugation. The binding site of NusE on NusB was determined using NMR chemical shift perturbation studies. We have also determined the NusE binding site in the ternary Escherichia coli NusB/NusE/boxA RNA complex and show that it is very similar to that in the NusB/NusE complex. There is one loop of residues (from 113 to 118 in NusB) affected by NusE binding in the ternary complex but not in the binary complex. This difference may be correlated to an increase in binding affinity of RNA for the NusB/NusE complex.

A 35-fold excess of methyl triflate (2) is required to quantitively prepare 3, the first phosphanyl phosphenium ion, from diphosphene 1. Experimental data and calculations indicate that the P=P bond becomes stronger upon alkylation.