Enzymatic activity and thermoresistance of improved microbial transglutaminase variants.

Microbial transglutaminase (MTG, EC 2.3.2.13) of Streptomyces mobaraensis is widely used in industry for its ability to synthesize isopeptide bonds between the proteinogenic side chains of glutamine and lysine. The activated wild-type enzyme irreversibly denatures at 60 °C with a pseudo-first-order kinetics and a half-life time (t1/2) of 2 min. To increase the thermoresistance of MTG for higher temperature applications, we generated 31 variants based on previous results obtained by random mutagenesis, DNA shuffling and saturation mutagenesis. The best variant TG16 with a specific combination of five of seven substitutions (S2P, S23Y, S24 N, H289Y, K294L) shows a 19-fold increased half-life at 60 °C (t1/2 = 38 min). As measured by differential scanning fluorimetry, the transition point of thermal unfolding was increased by 7.9 °C. Also for the thermoresistant variants, it was shown that inactivation process follows a pseudo-first-order reaction which is accompanied by irreversible aggregation and intramolecular self-crosslinking of the enzyme. Although the mutations are mostly located on the surface of the enzyme, kinetic constants determined with the standard substrate CBZ-Gln-Gly-OH revealed a decrease in KM from 8.6 mM (± 0.1) to 3.5 mM (± 0.1) for the recombinant wild-type MTG and TG16, respectively. The improved performance of TG16 at higher temperatures is exemplary demonstrated with the crosslinking of the substrate protein ß-casein at 60 °C. Using molecular dynamics simulations, it was shown that the increased thermoresistance is caused by a higher backbone rigidity as well as increased hydrophobic interactions and newly formed hydrogen bridges.

Transglutaminase: new insights into gelatin nanoparticle cross-linking.

Gelatin nanoparticles (GNPs) have demonstrated to be beneficial as a biodegradable and biocompatible delivery system. So far, nanoparticles prepared by the two-step desolvation technique were subsequently cross-linked by glutaraldehyde to guarantee storage stability. Although in vivo and in vitro toxicological studies have not revealed any glutaraldehyde related undesired effects, an alternative to chemical cross-linking could ease future clinical use in humans. Therefore, the recombinant enzyme microbial transglutaminase was used to examine its cross-linking abilities in nanoparticle production. Various process parameters, such as incubation time, temperature, medium, pH and the particle purification were evaluated regarding their impact on particle size and its distribution. Cross-linking reactions were best at 25°C using an ion-free solvent at a neutral pH and have been terminated after 12 h. Preliminary storage stability testing indicated adequate consistency of particle size and particle distribution making transglutaminase a potential candidate for glutaraldehyde substitution in future GNP production.

Validation of the PHEEM instrument in a Danish hospital setting.

The Postgraduate Hospital Educational Environment Measure (PHEEM) has been translated into Danish and then validated with good internal consistency by 342 Danish junior and senior hospital doctors. Four of the 40 items are culturally dependent in the Danish hospital setting. Factor analysis demonstrated that seven items are interconnected. This information can be used to shorten the instrument by perhaps another three items.

Carrier photogeneration, drift and recombination in a semiconducting carbon nanotube network.

Charge carrier photogeneration, drift and recombination in thin film networks of polymer-wrapped (6,5)-single-wall carbon nanotubes (SWNTs) blended with phenyl-C61-butyric acid methyl ester (PCBM) have been investigated by using transient photocurrent and time-delayed collection field (TDCF) techniques. Three distinct transient photocurrent components on the nano- and microsecond timescales have been identified. We attribute the dominant (>50% of total extracted charge) ultrashort photocurrent component with a decay time below our experimental time-resolution of 2 ns to the intratube hole motion. The second component on the few microsecond timescale is attributed to the intertube hole transfer, while the slowest component is assigned to the electron drift within the PCBM phase. The hole drift distance appears to be limited by gaps in the nanotube percolation network rather than by hole trapping or recombination. Photocurrent saturation was observed when excitation densities reached more than one charge pair per nanotube; we attribute this to the local electric field screening.

Below-gap excitation of semiconducting single-wall carbon nanotubes.

We investigate the optoelectronic properties of the semiconducting (6,5) species of single-walled carbon nanotubes by measuring ultrafast transient transmission changes with 20 fs time resolution. We demonstrate that photons with energy below the lowest exciton resonance efficiently lead to linear excitation of electronic states. This finding challenges the established picture of a vanishing optical absorption below the fundamental excitonic resonance. Our result points towards below-gap electronic states as an intrinsic property of semiconducting nanotubes.

Mutation spectrum in patients with Wiskott-Aldrich syndrome and X-linked thrombocytopenia: identification of twelve different mutations in the WASP gene.

Twelve different mutations in the WASP gene were found in twelve unrelated families with Wiskott-Aldrich syndrome (WAS) or X-linked thrombocytopenia (XLT). Four frameshift, one splice, one nonsense mutation, and one 18-base-pair deletion were detected in seven patients with WAS. Only missense mutations were found in five patients diagnosed as having XLT. One of the nucleotide substitutions in exon 2 (codon 86) results in an Arg to Cys replacement. Two other nucleotide substitutions in this codon, R86L and R86H, have been reported previously, both giving rise to typical WAS symptoms, indicating a mutational hot spot in this codon. The finding of mutations in the WASP gene in both WAS and XLT gives further evidence of these syndromes being allelic. The relatively small size of the WASP gene facilitates the detection of mutations and a reliable diagnosis of both carriers and affected fetuses in families with WAS or XLT.

Electron-phonon interaction in single-wall carbon nanotubes: A time-domain study

We investigate the electron-phonon (e-ph) interaction in single-wall carbon nanotube samples at room temperature using femtosecond time-resolved photoemission. By probing electrons from the vicinity of the Fermi level we are able to study the e-ph interaction in the metallic nanotube species only. The observed electron dynamics can be used to calculate e-ph scattering matrix elements for two likely scattering scenarios: forward scattering from twistons and backscattering by longitudinal acoustic phonons. The corresponding matrix elements reveal an intrinsically weak e-ph interaction approximately 50% smaller than predicted by tight-binding calculations.

Ultrafast dynamics of delocalized and localized electrons in carbon nanotubes.

We report on the dynamics of the dielectric function of single-wall carbon nanotubes in the 10-30 THz frequency range after ultrafast laser excitation. The absence of a distinct free-carrier response is attributed to the photogeneration of strongly bound excitons in the tubes with large energy gaps. We find a feature of enhanced transmission caused by the blocking of optical transitions in small-gap tubes. The rapid decay of a featureless background with pronounced dichroism is associated with the increased absorption of spatially localized charge carriers before thermalization is completed.

Anisotropy of quasiparticle lifetimes and the role of disorder in graphite from ultrafast time-resolved photoemission spectroscopy.

Femtosecond time-resolved photoemission of photoexcited electrons in highly oriented pyrolytic graphite (HOPG) provides strong evidence for anisotropies of quasiparticle (QP) lifetimes. Indicative of such anisotropies is a pronounced anomaly in the energy dependence of QP lifetimes between 1.1 and 1.5 eV--the vicinity of a saddle point in the graphite band structure. This is supported by recent ab initio calculations and a comparison with experiments on defect-enriched HOPG which reveal that disorder, e.g., defects or phonons, increases electron energy relaxation rates.

Serum levels of growth hormone binding protein in children with normal and precocious puberty: relation to age, gender, body composition and gonadal steroids.

AIM: To study the regulation of GHBP serum levels by gonadal steroids in normal and precocious puberty. STUDY PROTOCOL: We studied GHBP levels in relation to age, sex, pubertal maturation, body composition as well as to circulating IGF-I and gonadal steroid levels in 320 healthy children. Furthermore, we studied the regulation of circulating GHBP in 33 girls with central precocious puberty before and during gonadal suppression with GnRH agonist. METHODS: GHBP was determined by a time-resolved fluoroimmunoassay (GHBP TR-FIA) based on a commercially available immunoassay for GH, the DELFIA GH assay. RESULTS: In healthy children GHBP levels were significantly higher in normal girls compared with boys, and there was no significant increase in GHBP in puberty in both sexes. GHBP levels did not correlate with height (SDS), age, pubertal stage, IGF-I or testosterone/oestradiol levels in boys and girls, respectively. There were significant correlations between BMI and GHBP in boys and girls (R 2 = 0.14 and R 2 = 0.12, both P < 0.0001). Furthermore, GHBP correlated highly significantly with the percentage body fat, determined by BIA in 43 healthy girls (R 2 = 0. 40, P < 0.0001). GHBP levels were significantly higher in girls with central precocious puberty (CPP) (1.31 SDS (1.26), mean (SD)) compared to prepubertal controls (P < 0.0001), and above + 2 SD in 10 out of 33 patients. In girls with CPP, GHBP correlated inversely with oestradiol before treatment (R 2 = 0.26, P < 0.01) and there was a tendency towards a positive correlation with BMI (R 2 = 0.13, P = 0.078). By contrast, there were no signficant correlations between GHBP and IGF-I or height SDS. Gonadal suppression with GnRH agonist treatment caused a transient significant increase of 0.57 SD after 2 months of treatment (P < 0.001), but decreased to baseline levels hereafter. CONCLUSION: We conclude that in children, as in adults, body fat is the primary determinant for the circulating level of GHBP, and that the difference in body fat is probably the main factor for the higher levels of serum GHBP in girls compared with boys, as well as for the negative influence of testosterone levels in boys and of oestrogen levels in girls. The elevation in GHBP levels observed in girls with central precocious puberty is probably due their higher body fat content.

Movement characteristics of persons with prader-willi syndrome rising from supine.

PURPOSE: The purposes of this study were to: 1) determine if previously published descriptors of the supine to stand rising task in healthy individuals could be applied to the movements of persons with Prader-Willi Syndrome (PWS); and 2) assess upper extremity (UE), axial region (AX), and lower extremity (LE) movements among subjects with PWS compared with controls. METHODS: Nine subjects with PWS (seven-36 years of age) and matched controls were videotaped performing 10 rising trials. The UE, AX, and LE movements were classified using published descriptors. Occurrence frequencies of movement patterns, duration of movement, and the relationships among body region movement score, BMI, and age were determined. RESULTS: Subjects with PWS utilized developmentally less advanced asymmetrical rising patterns, took longer to rise, and demonstrated less within subject variability than controls. CONCLUSIONS: Categorical descriptors, with minor modifications, can be used to describe rising movements in persons with PWS. Knowledge of successful rising patterns may assist PTs when examining or planning intervention strategies for teaching the rising task.