Human thymidine kinase 2: molecular cloning and characterisation of the enzyme activity with antiviral and cytostatic nucleoside substrates

Based on amino acid sequence information from purified mitochondrial thymidine kinase (TK2), a cDNA of 1930 bp was cloned, containing an open reading frame encoding 232 amino acid residues starting with the N-terminal sequence determined from the native human protein preparation. Northern blot analysis with the cDNA coding region demonstrated several TK2 mRNAs, with 2 and 4 kb forms present in many tissues. We also characterised N-terminally truncated (starting at position 18) human TK2 with pharmacologically important antiviral and cytostatic nucleoside analogues. Results were highly similar to those with the native TK2 preparation. The anti-leukaemic drug arabinosyl cytosine is phosphorylated. The antitumour drug difluorodeoxycytidine and its metabolite difluorodeoxyuridine are good substrates, with K(m) values of 66 and 29 microM, respectively, and a relative Vmax of 0.6 compared to that of thymidine. Negative cooperativity was found with thymidine and the anti-HIV drug 3'-azidothymidine, but the reaction followed Michaelis-Menten kinetics with deoxycytidine, arabinosyl cytosine, and arabinosyl thymine. The results demonstrate a broad substrate specificity and complex kinetics, and suggest a role for TK2 in the activation of chemotherapeutic nucleoside analogues.     

Effect of the dynamic redistribution of impurities on the grain-boundary friction in nanocrystalline materials

A relaxation process involving the redistribution of impurity atoms along the grain boundaries in a nanocrystalline material under the action of a variable external load is considered. The frequency spectrum of internal friction in this system, which is found by solving the barodiffusion equation, has a peak shape. The intensity of this spectral peak is estimated.     

Spectral analysis of heart rate variability in patients with irritable bowel syndrome

BACKGROUND: Autonomic dysfunction, both adrenergic and cholinergic, has been associated with the irritable bowel syndrome (IBS). The accuracy of the methods in use, however, has been limited by the need for active co-operation by the patients, with consequent difficulties in standardization. The aim of this study was to investigate the function of the autonomic nervous system in patients with IBS by using spectral analysis of the heart rate variability, an accurate method depending very little on patient cooperation. METHODS: Eighteen patients with IBS were compared with 36 sex- and age-matched controls. Spectral analysis of heart rate variability was performed to quantify sympathetic and parasympathetic nerve activity. RESULTS: The patients with IBS had significantly higher sympathetic activity than controls (P = 0.005). There was no significant (P = 0.308) increase in parasympathetic activity. There were no significant differences in heart rate or blood pressure between the patients and controls. CONCLUSION: Spectral analysis of heart rate variability has been used to assess the function of the autonomic nervous system in patients with IBS. IBS patients have significantly increased symphathetic activity, whereas parasympathetic activity does not differ from that of controls.     

Lactate and pyruvate changes in the cerebral gray and white matter during posthypoxic seizures in newborn pigs

Cerebral lactate rises after chemically induced seizures, but it is not known if this occurs with posthypoxic seizures. We examined changes in lactate and pyruvate in gray and white matter in the newborn pig brain after a hypoxic insult known to produce seizures and permanent brain damage. Fourteen halothane-anesthetized piglets aged 24-49 h, were instrumented with a two-channel scalp EEG and microdialysis probes positioned in white and gray matter. Forty-five minutes of hypoxia were induced by reducing the fraction of inspired O2 to the maximum concentration at which EEG amplitude was < 7 microV. Postinsult EEG was classified as electroconvulsive activity (ECA) (n = 4) or burst suppression (n = 2), persistently low amplitude (n = 2), or intermittent spikes on normal background activity (n = 6). Six hours after the insult the brains were perfusion fixed for histologic probe localization. Plasma lactate and brain lactate had different time courses with brain having a persistently elevated lactate/pyruvate (L/P) ratio. The highest L/P ratios in gray and white matter were in the two pigs with persistently low amplitude EEG. There was no association between onset of electroconvulsive activity and an increase in lactate or L/P ratio. Posthypoxic energy metabolism is disturbed in both gray and white matter probably because of mitochondrial dysfunction. Seizure activity does not increase cerebral lactate or L/P ratio above the already raised levels found in posthypoxic encephalopathy. These findings cast further doubt on the hypothesis that such seizures are, in themselves, damaging.     

Texture formation on the friction surface in transformation-toughened ceramics

The surface of Y-TZP ceramics after dry friction on steel in a wide range of relative sliding velocities (0.2–11.1 m/s) was studied by X-ray diffraction. An analysis of the surface structure showed that the texture state of the friction surface is similar to that observed for analogous materials upon rough grinding. The main trends in the I ₍₀₀₂₎/I ₍₂₀₀₎ intensity ratio characterizing the tetragonal ZrO₂ phase as a function of the sliding velocity and the ceramic grain size are established. The mechanisms of lattice reorientation operative at various sliding velocities are discussed.     

Characterisation of anthocyanins in red cabbage using high resolution liquid chromatography coupled with photodiode array detection and electrospray ionization-linear ion trap mass spectrometry

The aim of this work was to analyse and tentatively identify anthocyanin species in red cabbage using HPLC/DAD-ESI/Qtrap MS. The extraction was realized by using a pressurized liquid technique and the separation of the pigments was achieved by a high resolution liquid chromatography system with a 1.8 μm particles C-18 column. Photodiode array detection was employed to determine the UV/Vis spectral characteristic of the pigments. Electrospray ionization-linear ion trap mass spectrometry allowed the specific determination of the fragmentation patterns of the anthocyanins, by performing different ion scan modes. Twenty four anthocyanins were separated and identified, all having cyanidin as aglycon, represented as mono- and/or di-glycoside, and acylated, or not, with aromatic and aliphatic acids. Nine anthocyanins were identified for the first time in red cabbage.     

Cysteinyl and substrate radical formation in active site mutant E441Q of Escherichia coli class I ribonucleotide reductase

All classes of ribonucleotide reductase are proposed to have a common reaction mechanism involving a transient cysteine thiyl radical that initiates catalysis by abstracting the 3'-hydrogen atom of the substrate nucleotide. In the class Ia ribonucleotide reductase system of Escherichia coli, we recently trapped two kinetically coupled transient radicals in a reaction involving the engineered E441Q R1 protein, wild-type R2 protein, and substrate (Persson, A. L., Eriksson, M., Katterle, B., P?tsch, S., Sahlin, M., and Sj?berg, B.-M. (1997) J. Biol. Chem. 272, 31533-31541). Using isotopically labeled R1 protein or substrate, we now demonstrate that the early radical intermediate is a cysteinyl radical, possibly in weak magnetic interaction with the diiron site of protein R2, and that the second radical intermediate is a carbon-centered substrate radical with hyperfine coupling to two almost identical protons. This is the first report of a cysteinyl free radical in ribonucleotide reductase that is a kinetically coupled precursor of an identified substrate radical. We suggest that the cysteinyl radical is localized to the active site residue, Cys439, which is conserved in all classes of ribonucleotide reductase, and which, in the three-dimensional structure of protein R1, is positioned to abstract the 3'-hydrogen atom of the substrate. We also suggest that the substrate radical is localized to the 3'-position of the ribose moiety, the first substrate radical intermediate in the postulated reaction mechanism.