Identification of the nitrogen split interstitial (N-N)(N) in GaN.

Combining electron paramagnetic resonance, density functional theory, and positron annihilation spectroscopy (PAS), we identify the nitrogen interstitial defect in GaN. The isolated interstitial is unstable and transforms into a split interstitial configuration (N-N)(N). It is generated by particle irradiation with an introduction rate of a primary defect, pins the Fermi level at E(C)-1.0 eV for high fluences, and anneals out at 400 °C. The associated defect, the nitrogen vacancy, is observed by PAS only in the initial stage of irradiation.

Effects of psychological stress on the cerebral processing of visceral stimuli in healthy women.

The aim of the study was to analyse effects of psychological stress on the neural processing of visceral stimuli in healthy women. The brain functional magnetic resonance imaging blood oxygen level-dependent response to non-painful and painful rectal distensions was recorded from 14 healthy women during acute psychological stress and a control condition. Acute stress was induced with a modified public speaking stress paradigm. State anxiety was assessed with the State-Trait-Anxiety Inventory; chronic stress was measured with the Perceived Stress Questionnaire. During non-painful distensions, activation was observed in the right posterior insular cortex (IC) and right S1. Painful stimuli revealed activation of the bilateral anterior IC, right S1, and right pregenual anterior cingulate cortex. Chronic stress score was correlated with activation of the bilateral amygdala, right posterior IC (post-IC), left periaqueductal grey (PAG), and right dorsal posterior cingulate gyrus (dPCC) during non-painful stimulation, and with activation of the right post-IC, right PAG, left thalamus (THA), and right dPCC during painful distensions. During acute stress, state anxiety was significantly higher and the acute stress - control contrast revealed activation of the right dPCC, left THA and right S1 during painful stimulation. This is the first study to demonstrate effects of acute stress on cerebral activation patterns during visceral pain in healthy women. Together with our finding that chronic stress was correlated wit the neural response to visceral stimuli, these results provide a framework for further studies addressing the role of chronic stress and emotional disturbances in the pathophysiology of visceral hyperalgesia.

Rate-limiting reactions determining different activation kinetics of Kv1.2 and Kv2.1 channels.

To identify the mechanisms underlying the faster activation kinetics in Kv1.2 channels compared to Kv2.1 channels, ionic and gating currents were studied in rat Kv1.2 and human Kv2.1 channels heterologously expressed in mammalian cells. At all voltages the time course of the ionic currents could be described by an initial sigmoidal and a subsequent exponential component and both components were faster in Kv1.2 than in Kv2.1 channels. In Kv1.2 channels, the activation time course was more sigmoid at more depolarized potentials, whereas in Kv2.1 channels it was somewhat less sigmoid at more depolarized potentials. In contrast to the ionic currents, the ON gating currents were similarly fast for both channels. The main portion of the measured ON gating charge moved before the ionic currents were activated. The equivalent gating charge of Kv1.2 ionic currents was twice that of Kv2.1 ionic currents, whereas that of Kv1.2 ON gating currents was smaller than that of Kv2.1 ON gating currents. In conclusion, the different activation kinetics of Kv1.2 and Kv2.1 channels are caused by rate-limiting reactions that follow the charge movement recorded from the gating currents. In Kv1.2 channels, the reaction coupling the voltage-sensor movement to the pore opening contributes to rate limitation in a voltage-dependent fashion, whereas in Kv2.1 channels, activation is additionally rate-limited by a slow reaction in the subunit gating.

Molecular regions controlling the activity of CNG channels.

The alpha subunits of CNG channels of retinal photoreceptors (rod) and olfactory neurons (olf) are proteins that consist of a cytoplasmic NH(2) terminus, a transmembrane core region (including the segments S1-S6), and a cytoplasmic COOH terminus. The COOH terminus contains a cyclic nucleotide monophosphate binding domain NBD) that is linked by the C-linker (CL) to the core region. The binding of cyclic nucleotides to the NBD promotes channel opening by an allosteric mechanism. We examined why the sensitivity to cGMP is 22 times higher in olf than in rod by constructing chimeric channels and determining the [cGMP] causing half maximum channel activity (EC(50)). The characteristic difference in the EC(50) value between rod and olf was introduced by the NH(2) terminus and the core-CL region, whereas the NBD showed a paradoxical effect. The difference of the free energy difference Delta(DeltaG) was determined for each of these three regions with all possible combinations of the other two regions. For rod regions with respect to corresponding olf regions, the open channel conformation was destabilized by the NH(2) terminus (Delta(DeltaG) = -1.0 to -2.0 RT) and the core-CL region (Delta(DeltaG) = -2.0 to -2.9 RT), whereas it was stabilized by the NBD (Delta(DeltaG) = 0.3 to 1.1 RT). The NH(2) terminus deletion mutants of rod and olf differed by Delta(DeltaG) of only 0.9 RT, whereas the wild-type channels differed by the much larger value of 3.1 RT. The results show that in rod and olf, the NH(2) terminus, the core-CL region, and the NBD differ by characteristic Delta(DeltaG) values that do not depend on the specific composition of the other two regions and that the NH(2) terminus generates the main portion of Delta(DeltaG) between the wild-type channels.

Role of the S2 and S3 segment in determining the activation kinetics in Kv2.1 channels.

We constructed chimeras between the rapidly activating Kv1.2 channel and the slowly activating Kv2.1 channel in order to study to what extent sequence differences within the S1-S4 region contribute to the difference in activation kinetics. The channels were expressed in Xenopus oocytes and the currents were measured with a two-microelectrode voltage-clamp technique. Substitution of the S1-S4 region of Kv2.1 subunits by the ones of Kv1.2 resulted in chimeric channels which activated more rapidly than Kv2.1. Furthermore, activation kinetics were nearly voltage-independent in contrast to the pronounced voltage-dependent activation kinetics of both parent channels. Systematic screening of the S1-S4 region by the replacement of smaller protein parts resolved that the main functional changes generated by the S1-S4 substitution were generated by the S2 and the S3 segment. However, the effects of these segments were different: The S3 substitution reduced the effective gating charge and accelerated both a voltage-dependent and a voltage-independent component of the activation time course. In contrast, the S2 substitution accelerated predominantly the voltage-dependent component of the activation time course thereby leaving the effective gating charge unchanged. It is concluded that the S2 and the S3 segment determine the activation kinetics in a specific manner.

Structural elements determining activation kinetics in Kv2.1.

Voltage-dependent K+ channels open when depolarizing the membrane voltage. Among the different alpha-subunits, the time course of current activation spreads over a wide range. The structural basis underlying this diversity is not known. We constructed multiple chimeras between two voltage-dependent K+ channels, the rapidly activating Kv1.2 and the slowly activating Kv2.1, and we focused on the C-terminal half of the core region. The general strategy was to substitute parts of Kv2.1 by corresponding parts of Kv1.2 and to test for an acceleration of activation. We identified three regions which contribute to the determination of the activation kinetics: the S5-pore linker, the deep pore, and the S4-segment.

Deletion mutants of the Escherichia coli K-12 mannitol permease: dissection of transport-phosphorylation, phospho-exchange, and mannitol-binding activities.

We have constructed a series of deletion mutations of the cloned Escherichia coli K-12 mtlA gene, which encodes the mannitol-specific enzyme II of the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system. This membrane-bound permease consists of 637 amino acid residues and is responsible for the concomitant transport and phosphorylation of D-mannitol in E. coli. Deletions into the 3' end of mtlA were constructed by exonuclease III digestion. Restriction mapping of the resultant plasmids identified several classes of deletions that lacked approximately 5% to more than 75% of the gene. Immunoblotting experiments revealed that many of these plasmids expressed proteins within the size range predicted by the restriction analyses, and all of these proteins were membrane localized, which demonstrated that none of the C-terminal half of the permease is required for membrane insertion. Functional analyses of the deletion proteins, expressed in an E. coli strain deleted for the chromosomal copy of mtlA, showed that all but one of the strains containing confirmed deletions were inactive in transport and PEP-dependent phosphorylation of mannitol, but deletions removing up to at least 117 amino acid residues from the C terminus of the permease were still active in catalyzing phospho exchange between mannitol 1-phosphate and mannitol. A deletion protein that lacked 240 residues from the C terminus of the permease was inactive in phospho exchange but still bound mannitol with high affinity. These experiments localize sites important for transport and PEP-dependent phosphorylation to the extreme C terminus of the mannitol permease, sites important for phospho exchange to between residues 377 and 519, and sites necessary for mannitol binding to the N-terminal 60% of the molecule. The results are discussed with respect to the fact that the mannitol permease consists of structurally independent N- and C-terminal domains.

Plasmid vector for overproduction and export of recombinant protein in Escherichia coli: efficient one-step purification of a recombinant antigen from Echinococcus multilocularis (Cestoda).

We describe the use of the Escherichia coli plasmid vector, pVB2, for high-level expression and export of recombinant protein. The pBR322 derivative pVB2 harbors the mglB gene, which codes for the galactose-binding protein (GBP) of E. coli. GBP is exported into the periplasmic space of the bacterial cell. Gene mglB contains an EcoRI restriction site close to its 3' end which allows simple in-frame insertion of EcoRI fragments obtained from recombinant lambda gt11 phages. The pVB2 vector was used to express an antigen from Echinococcus multilocularis. The recombinant protein amounted to over 50% of total cellular protein and could be efficiently isolated from the periplasm by osmotic shock. The application of the purified antigen in an ELISA enabled a clear and specific detection of anti-Ec. multilocularis antibodies in human patients' sera, which had been immunosorbed with a periplasmic extract (containing wt GBP) before investigation. These data show the general usefulness of pVB2 as an expression vector for producing in E. coli diagnostically relevant antigens from any infective organism.

Sequence of the mglB gene from Escherichia coli K12: comparison of wild-type and mutant galactose chemoreceptors.

The mglB gene of Escherichia coli codes for a galactose-binding protein (GBP) that serves both as the galactose chemoreceptor and as the recognition component of the beta-methylgalactoside transport system. The mglB551 mutation eliminates the chemotactic function of GBP without altering its transport or substrate-binding properties. To investigate the interaction between GBP and Trg, the chemotactic signal transducer for galactose, we sequenced the mglB genes from wild-type and mglB551 mutant strains. The mutation causes the replacement of Gly74 of GBP by Asp. This residue is located in alpha-Helix III at the tip of the P domain in the GBP tertiary structure farthest removed from the substrate-binding cleft between the P and Q domains. We conclude that Helix III must be part of, or at least adjacent to, the recognition site for Trg. Our sequence also included part of the mglA gene, which is immediately distal to mglB. The amino acid sequence deduced for the beginning of the MglA protein showed homology with a family of polypeptides that contain an ATP-binding site and are components of binding-protein-dependent transport systems.