Mice with defects in HB-EGF ectodomain shedding show severe developmental abnormalities

Heparin-binding EGF-like growth factor (HB-EGF) is first synthesized as a membrane-anchored form (proHB-EGF), and its soluble form (sHB-EGF) is released by ectodomain shedding from proHB-EGF. To examine the significance of proHB-EGF processing in vivo, we generated mutant mice by targeted gene replacement, expressing either an uncleavable form (HBuc) or a transmembrane domain-truncated form (HBdeltatm) of the molecule. HB(uc/uc) mice developed severe heart failure and enlarged heart valves, phenotypes similar to those in proHB-EGF null mice. On the other hand, mice carrying HBdeltatm exhibited severe hyperplasia in both skin and heart. These results indicate that ectodomain shedding of proHB-EGF is essential for HB-EGF function in vivo, and that this process requires strict control.     

Dynamics of the protein structure of T169S pyranose 2‐oxidase in solution: Molecular dynamics simulation

Pyranose 2‐oxidase (P2O) from Trametes multicolor contains FAD as cofactor, and forms a tetramer. The protein structure of a mutated P2O, T169S (Thr169 is replaced by Ser), in solution was studied by means of molecular dynamics simulation and analyses of photoinduced electron transfer (ET) from Trp168 to excited isoalloxazine (Iso*), and was compared with wild type (WT) P2O. Hydrogen bonding between Iso and nearby amino acids was very similar as between T169S and WT protein. Distances between Iso and Tyr456 were extremely heterogeneous among the subunits, 1.7 (1.5 in WT) in subunit A (Sub A), 0.97 (2.2 in WT) in Sub B, 1.3 (2.1 in WT) in Sub C, 1.3 nm (2.0 in WT) in Sub D. Mean values of root of mean square fluctuation over all residues were greater by four times than those in WT. This suggests that the protein structure of T169S is much more flexible than that of WT. Electrostatic (ES) energies between Iso anion in one subunit and ionic groups in the entire protein were evaluated. It was found that more than 50% of the total ES energy in each subunit is contributed from other subunits. Reported fluorescence decays were analyzed by a method as WT, previously reported. Electron affinities of Iso* in T169S were appreciably higher than those in WT. Static dielectric constants near Iso and Trp168 were also quite higher in T169S than those in WT.     

Hydrogen Cyanide Production due to Mid-Size Impacts in a Redox-Neutral N2-Rich Atmosphere

Cyanide compounds are amongst the most important molecules of the origin of life. Here, we demonstrate the importance of mid-size (0.1–1 km in diameter) hence frequent meteoritic impacts to the cyanide inventory on the early Earth. Subsequent aerodynamic ablation and chemical reactions with the ambient atmosphere after oblique impacts were investigated by both impact and laser experiments. A polycarbonate projectile and graphite were used as laboratory analogs of meteoritic organic matter. Spectroscopic observations of impact-generated ablation vapors show that laser irradiation to graphite within an N₂-rich gas can produce a thermodynamic environment similar to that produced by oblique impacts. Thus, laser ablation was used to investigate the final chemical products after this aerodynamic process. We found that a significant fraction (>0.1 mol%) of the vaporized carbon is converted to HCN and cyanide condensates, even when the ambient gas contains as much as a few hundred mbar of CO₂. As such, the column density of cyanides after carbon-rich meteoritic impacts with diameters of 600 m would reach ~10 mol/m² over ~10² km² under early Earth conditions. Such a temporally and spatially concentrated supply of cyanides may have played an important role in the origin of life.     

Decoupling Analysis of Four Selected Countries

We examine decoupling conditions of domestic extraction of materials, energy use, and sulfur dioxide (SO₂) emissions from gross domestic product (GDP) for two BRIC (Brazil, Russia, India and China) countries (i.e., China and Russia) and two Organisation for Economic Co‐operation and Development (OECD) countries (Japan and the United States) during 2000–2007, using a pair of decoupling indicators for resource use (D_r) and waste emissions (D_e) and the decoupling chart, which can distinguish between absolute decoupling, relative decoupling, and non‐decoupling. We find that (1) during 2000–2007, decoupling between environmental indicators and GDP was higher in the two OECD countries as compared with the two BRIC countries. The key reason is that these countries were in different development stages with different economic growth rates. (2) Changes in environmental policies can significantly influence the degree of decoupling in a country. (3) China, Japan, and the United States were more successful in decoupling SO₂ emissions from GDP than in decoupling material and energy use from GDP. The main reason is that, unlike resource use, waste emissions (e.g., SO₂ emissions) can be reduced by effective end‐of‐pipe treatment. (4) The decoupling indicator is different from the changing rate of resource use and waste emissions. If two countries have different GDP growth rates, even though they may have similar values using the decoupling indicator, they may show different rates of change for resource use and waste emissions.     

Mutant luciferase enzymes from fireflies with increased resistance to benzalkonium chloride

Benzalkonium chloride (BAC), used to extract intracellular ATP, interferes with subsequent firefly luciferase-luciferin assays. There was a significant difference among wild-type luciferases with respect to BAC resistance. Luciola lateralis luciferase (LlL) was the most tolerant, followed by Luciola cruciata luciferase (LcL) and Photinus pyralis luciferase. Random mutagenesis of thermostable mutants of LcL showed that the Glu490Lys mutation contributes to improved resistance to BAC. The corresponding Glu490Lys mutation was introduced into thermostable mutants of LlL by site-directed mutagenesis. Kinetic analysis demonstrated that the resultant LlL-217L490K mutant, having both an Ala217Leu and a Glu490Lys mutation, showed the highest resistance to BAC, with an initial remaining bioluminescence intensity of 87.4% and a decay rate per minute of 29.6% in the presence of 0.1% BAC. The Glu490Lys mutation was responsible for increased resistance to inactivation but not inhibition by BAC. The LlL-217L490K had identical thermostability and pH stability to the parental thermostable mutant. From these results, it was concluded that the LlL-217L490K enzyme is advantageous for hygiene monitoring and biomass assays based on the ATP-bioluminescence methodology. This is the first report demonstrating improved resistance to BAC of the firefly luciferase enzyme.     

43Ca NMR studies of Ca2+-Tetrahymena calmodulin complexes

⁴³Ca NMR spectra of Ca²⁺-Tetrahymena calmodulin(Tet. CaM.) complexes have been observed under various conditions. Off-rate of Ca²⁺ from Tet. CaM. is estimated to be approx. 2.7 × 10³ s^?1 under a certain assumption. Relaxation rates of ⁴³Ca NMR of Ca²⁺-Tet. CaM. are remarkably increased(by one order in magnitude) by adding trifluoperazine(TFP), a potent calmodulin antagonist. Relaxation parameters estimated suggest that Ca²⁺ mobility is reduced by the TFP binding. A stoichiometry of TFP is two moles per Tet. CaM. molecule. The relaxation rates of ⁴³Ca NMR signals are increased by adding excessive Mg²⁺ to the Ca²⁺-Tet. CaM. solutions. The addition of Mg²⁺ to the Ca²⁺-Tet. CaM. complex decreases apparent pKa value of the complex as well.     

Normalization of phospholipids concentration of the gastric mucosa was observed in patients with peptic ulcer after eradication of Helicobacter pylori

Background. Phospholipids concentration in the gastric mucosa decreased in patients with Helicobacter pylori infection. The aim of this study is to examine the effects of eradication of H. pylori on decreasing the phospholipids concentration in the gastric mucosa in patients with gastric or duodenal ulcer. Materials and Methods. Phospholipids (phosphatidylcholine, phosphatidylethanolamine, and sphingonomyeline) were measured in biopsy specimens from the antrum and corpus using thin‐layer chromatography. In H. pylori positive patients with gastric ulcer (n = 26) and duodenal ulcer (n = 13), and H. pylori negative controls (n = 20), the biopsy specimens were obtained before and 3 months after eradication. Eradication was performed using lansoprazole, amoxycillin, and clarithromycin. Results. Compared with the H. pylori negative control group, the concentrations of phosphatidylcholine and phosphatidylethanolamine decreased significantly in the gastric ulcer group in both antrum and corpus mucosa, and in the duodenal ulcer group in antrum mucosa. This decrease returned to the control level after eradication. Conclusions. This study demonstrates that the eradication of H. pylori in patients with peptic ulcer normalized the decrease of phosphatidylcholine and phosphatidylethanolamine in the gastric mucosa.     

Characteristics of hepatitis B virus isolates of genotype G and their phylogenetic differences from the other six genotypes (A through F)

Eight hepatitis B virus (HBV) isolates of genotype G were recovered from patients and sequenced over the entire genome. Six of them had a genomic length of 3,248 bp and two had genomic lengths of 3,239 bp (USG15) and 3,113 bp (USG18) due to deletions. The 10 HBV/G isolates, including the 8 sequenced isolates as well as the original isolate (AF160501) and another isolate (B1-89), had a close sequence homology of 99.3 to 99.8% among themselves (excluding USG18 with a long deletion) but of <88.7% to any of the 68 HBV isolates of the other six genotypes with the full-length sequence known. The eight HBV/G isolates possessed an insertion of 36 bp in the core gene and two stop codons in the precore region, as did the AF160501 and B1-89 isolates. The 10 HBV/G isolates clustered on a branch separate from those bearing the other six genotypes (A through F [A-F]) in the phylogenetic tree constructed from full-length sequences of 78 HBV isolates as well as in those constructed from the core, polymerase, X, and envelope genes. Despite two stop codons in the precore region that prohibited the translation of the HBV e antigen (HBeAg), all of the eight patients with HBV/G infection possessed the HBeAg in serum. By restriction fragment length polymorphism of the surface gene, all of the eight patients were found to be coinfected with HBV of genotype A (HBV/A), which would be responsible for the expression of HBeAg in them. It is worthy of examination to determine how coinfection occurs and whether HBV/G needs HBV/A for replication.