Preparation of defined molecular species of lactosylceramide by chemical deacylation and reacylation withN-succinimidyl fatty acid esters

A procedure for the preparation of specific molecular species ofd-erythro-lactosylceramide involving deacylation and reacylation of lactosylceramide prepared from bovine brain gangliosides is described. Lactosylceramide wasN-deacylated by alkaline hydrolysis and the resulting four lysolactosylceramides, which contained d18∶1, d20∶1, d18∶0 and d20∶0 long-chain bases, were simultaneously re-N-acylated with theN-succinimidyl ester of either 16∶0, 18∶0, 20∶0, 24∶0, 20∶1, 22∶1 or 24∶1 fatty acid. The resulting lactosylceramide contained four molecular species of lactosylceramides, i.e., d18∶1, d20∶1, d18∶0 and d20∶0 long-chain bases coupled with the fatty acid that was introduced. Lactosylceramides prepared in this manner were separated into four individual molecular species by high-performance liquid chromatography (HPLC). Each of the purified molecular species of lactosylceramide was quantitated by HPLC after derivatization with benzoylchloride and was characterized by mass spectrometry. The yields of reacylated lactosylceramide were 38–58% relative to the starting lactosylceramide; the purity of each of the molecular species of lactosylceramide was greater than 95%. The glycosphingolipid nomenclature is as recommended by the IUPAC-IUB Commission on Biochemical Nomenclature (1). GalCer, galactosylceramide, Gal(β1-1)Cer; GlcCer, glucosylceramide, Glc(β1-1)Cer; LacCer, lactosylceramide, Gal(β1-4)GlcCer; GbOse₃Cer, globotriaosylceramide, Gal(α1-4)Gal(β1-4)GlcCer; GbOse₄Cer, globotetraosylceramide, GalNAc(β1-3)Gal(α1-4)-Gal(β1-4)GlcCer; GgOse₃Cer, gangliotriaosylceramide, GalNAc-(β1-4)Gal(β1-4)GlcCer; GgOse₄Cer, gangliotetraosylceramide, Gal(β1-3)GalNAc(β1-4)Gal(β1-4)GlcCer; GM3, (NeuAcα2-3)-Galβ1-4GlcCer; GM1, Galβ1-3GalNAcβ1-4(NeuAcα2-3)Galβ1-4-GlcCer. The molecular species abbreviations suggested by Breimeret al. (2) are used. For example, in the notation d18∶1−18∶0, the d18∶1 represents the long-chain base sphingosine (d-erythro-1,3-dihydroxy-2-amino-trans-4-octadecene) and 18∶0 represents the fatty acid (octadecanoic acid).     

Influence of soil physical properties, fertiliser type and moisture tension on N2O and NO emissions from nearly saturated Japanese upland soils

In Japan, upland soils are an important source of nitrous oxide (N₂O) and nitric oxide (NO) gas emissions. This paper reports on an investigation of the effect of soil moisture near saturation on N₂O and NO emission rates from four upland soils in Japan of contrasting texture. The aim was to relate these effects to soil physical properties. Intact cores of each soil type were incubated in the laboratory at different moisture tensions after fertilisation with NH₄-N, NO₃-N or zero N. Emissions of N₂O and NO were measured regularly over a 16–20 day period. At the end of the incubation, soil cores were analysed for physical properties. Moisture and N fertiliser significantly affected rates of emissions of both N₂O and NO with large differences between the soil types. Nitrous oxide emissions were greatest in the finer-textured soils, whereas NO emissions were greater in the coarser-textured soils. Emissions of N₂O increased at higher moisture contents in all soils, but the magnitude of increase was much greater in finer-textured soils. Nitric oxide emissions were only significant in soils fertilised with NH₄-N and were negatively correlated with soil moisture. Analysis of soil properties showed that there was a strong relationship between the magnitude of emissions and soil physical properties. The importance of soil wetness to gas emissions was mainly through its influence on soil air-filled porosity, which itself was related to gas diffusivity. From the results of this research, we can now estimate likely effects of soil texture on emissions through the influence of soil type on soil aeration and soil drainage. This is of particular value in modelling N₂O and NO emissions from soil moisture status and land use inputs.     

Point mutation of alanine (31) to valine prohibits the folding of reduced lysozyme by sulfhydryl--disulfide interchange

In the preceding paper in this issue, we described the overproduction of one mutant chicken lysozyme in Escherichia coil.Since this lysozyme contained two amino acid substitutions (Ala³¹ValandAsn¹⁰⁶Ser)in addition to an extra methionine residue at theNH₂-terminus the substituted amino acid residues were convertedback to the original ones by means of oligonucleotide-directedsite-specific mutagenesis and in vitro recombination. Thus fourkinds of chicken lysozyme [Met^–1 Val³¹Ser¹⁰⁶-, Met^–1Ser¹⁰⁶-,Met^–1 Val³¹-and Met^–1 (wild type)] wereexpressed in E. coli. From the results of folding experimentsof the reduced lysozymes by sulfhydryl-disulfide interchangeat pH 8.0 and 38°C, follow ed by the specific activity measurementsof the folded en zymes, the following conclusions can be drawn:(i) an extra methionine residue at the NH₂-terminus reducesthe folding rate but does not affect the lysozyme activity ofthe folded enzyme; (ii) the substitution of Asn¹⁰⁶ by Ser decreasesthe activity to 58% of that of intact native lysozyme withoutchanging the folding rate; and (iii) the substitution of Ala³¹Val prohibits the correct folding of lysozyme. Since the wildtype enzyme (Met^–1-lysozyme) was activated in vitro withoutloss of specific activity, the systems described in this study(mutagenesis, overproduction, purification and folding of inactivemutant lysozymes) may be useful in the study of folding pathways,expression of biological activity and stability of lysozyme.     

Hydrogen concentration in water from an Alkali–Ion–Water electrolyzer having a platinum-electroplated titanium electrode

The supersaturated concentration of hydrogen in electrolyzed water obtained from a flow-type electrolytic cell was studied under various electrolysis conditions. The degree of supersaturation was found to decrease as the solution supply rate to the cell increased. The ratio of observed hydrogen concentration to the theoretical hydrogen concentration obtained from the electrochemical equivalent, as calculated from the transfer of charge in the cell, was found to increase with the solution supply rate. The concentration of hydrogen in solution has a maximum at a current density of approximately 0.3 A dm^–2. This maximum was found to be independent of the flow rate, indicating that the hydrogen concentration is related to both the diffusion of dissolved hydrogen from the electrode surface to the bulk solution and hydrogen bubble growth.     

How does heme axial ligand deletion affect the structure and the function of cytochrome b562?

We have recently generated a new mutant of cytochrome b₅₆₂ (cytb₅₆₂)in which Met7, one of the axial heme ligands, is replaced byAla (M7A cytb₅₆₂). The M7A cytb₅₆₂ can bind heme and the UV-visibleabsorption spectrum is of a typical high-spin ferric heme. Toinvestigate the effect of the lack of Met7 ligation on the structuralintegrity of cytb₅₆₂, thermal transition analyses of M7A cytb₅₆₂were conducted. From the thermodynamic parameters obtained,it is concluded that the folding of M7A cytb₅₆₂ is comparableto the apoprotein despite the presence of heme. On the otherhand, exogenous ligands such as cyanide and azide ions are readilybound to the heme iron, indicating that the axial coordinationsite is available for substrate binding. The peroxidase activityof this mutant is thus examined to evaluate new enzymatic functionat this site and M7A cytb₅₆₂ was found to catalyze an oxidationreaction of aromatic substrates with hydrogen peroxide. Theseobservations demonstrate that the Met7/His102 bis-ligation tothe heme iron is crucial for the stable folding of cytb₅₆₂,whereas the functional conversion of cytb₅₆₂ is successfullyachieved by the loose folding together with the open coordinationsite.     

Ethanol-tolerant Saccharomyces cerevisiae strains isolated under selective conditions by over-expression of a proofreading-deficient DNA polymerase δ

Ethanol damages the cell membrane and functional proteins, gradually reducing cell viability, and leading to cell death during fermentation which impairs effective bioethanol production by budding yeast Saccharomyces cerevisiae. To obtain more suitable strains for bioethanol production and to gain a better understanding of ethanol tolerance, ethanol-tolerant mutants were isolated using the novel mutagenesis technique based on the disparity theory of evolution. According to this theory evolution can be accelerated by affecting the lagging-strand synthesis in which DNA polymerase δ is involved. Expression of the pol3-01 gene, a proofreading-deficient of DNA polymerase δ, in S. cerevisiae W303-1A grown under conditions of increasing ethanol concentration resulted in three ethanol-tolerant mutants (YFY1, YFY2 and YFY3), which could grow in medium containing 13% ethanol. Ethanol productivity also increased in YFY strains compared to the wild-type strain in medium containing 25% glucose. Cell morphology of YFY strain cells was normal even in the presence of 8% ethanol, whereas W303-1A cells were expanded by a big vacuole. Furthermore, two of these mutants were also resistant to high-temperature, Calcofluor white and NaCl. Expression levels of TPS1 and TSL1, which are responsible for trehalose biosynthesis, were higher in YFY strains relative to W303-1A, resulting in high levels of intracellular trehalose in YFY strains. This contributed to the multiple-stress tolerance that makes YFY strains suitable for the production of bioethanol.     

Cloning and characterization of a novel phytase from wastewater treatment yeast Hansenula fabianii J640 and expression in Pichia pastoris

Phosphohydrolysis of organic phosphorus compounds by acid phosphatases (EC 3.1.3.1 and EC 3.1.3.2) is an important method for efficient removal of phosphorus from high concentration organic wastewater. Another important method is supplementation of animal feed with phytase (EC 3.1.3.8 and EC 3.1.3.26), which improves the availability of phytate-phosphates (phosphate that are hydrolyzed by phytases), making it possible to add less phosphate to animal feed and resulting in the excretion of less phosphorus by the animals. In the present study, we purified a novel phytase from the wastewater treatment yeast Hansenula fabianii J640 (Hfphytase), cloned the 1456 bp open reading frame (ORF) encoding Hfphytase, and characterized Hfphytase. The molecular weight of Hfphytase after deglycosylation by PNGaseF was 49 kDa. The optimal pH and temperature for enzyme activity were 4.5 and 50 °C, respectively. Hfphytase exhibits 40% identity with Debaryomyces castellii phytase, 37% identity with Aspergillus niger PhyB, and 34% identity with Saccharomyces cerevisiae Pho5p. Recombinant Hfphytase was transformed and expressed in Pichia pastoris. The yield was 23 g/l by jar fermenter cultivation. The marked phosphohydrolysis activity exhibited by Hfphytase on six substrates (pNP-P, sodium phytate, glucose-1 phosphate, glucose-6 phosphate, α-glycerophosphate and β-glycerophosphate) indicated that it is a non-specific acid phosphatase.     

Development of training method for vessel traffic service based on cognitive process

Cognition, Technology & Work - Vessel traffic service (VTS) is important to protect the safety of maritime traffic. Along with the expansion of monitoring area per VTS operator in Tokyo Bay,...