A novel method for measuring human hepatic lipase activity in postheparin plasma

金沢大学医薬保健研究域医学系The objective of this study was to establish a hepatic lipase (HL) assay method that can be applied to automatic clinical analyzers. Seventy-four hyperlipidemic subjects (men/women 45/29) were recruited. Lipase activity was assayed measuring the increase in absorbance at 546 nm due to quinonediimine dye production. Reaction mixture R-1 contained 50 mM Tris-HCl (pH 9.5), 0.5 mM glycerol-1,2-dioleate, 0.4% (unless otherwise noted) polyoxyethylenenonylphenylether, 3 mM ATP, 3 mM MgCl2, 1.5 mM CaCl2, monoacylglycerol-specific lipase, glycerol kinase, glycerol-3-phosphate oxidase, 0.075% N,N-bis-(4-sulfobutyl)-3-methylaniline-2 Na, peroxidase, ascorbic acid oxidase. Reaction mixture R-2 contained 50 mM Tris-HCl (pH9.5), 0.15% 4-aminoantypirine. Automated assay for activity was performed with a Model 7080 Hitachi analyzer. In the lipase assay, 160 μl of R-1 was incubated at 37°C with 3 μl of samples for 5 min, and 80 μl of R-2 was added. Within-run coefficient of variations was 0.9-1.0%. Calibration curve of lipase activity was linear (r = 0.999) between 0 and 320 U/l. Analytical recoveries of purified HL added to plasma were 96.6-99.8%. HL activity in postheparin plasma measured in this method had a closer correlation with HL mass by a sandwich ELISA (r = 0.888, P , 0.0001) than those in the conventional method using [ 14C-]triolein (r = 0.730, P < 0.0001). This assay method for HL activity can be applied to an automatic clinical analyzer. Copyright © 2007 by the American Society for Biochemistry and Molecular Biology, Inc

Characterization of an Alkali- and Halide-Resistant Laccase Expressed in E. coli: CotA from <i>Bacillus clausii</i>

The limitations of fungal laccases at higher pH and salt concentrations have intensified the search for new extremophilic bacterial laccases. We report the cloning, expression, and characterization of the bacterial cotA from Bacillus clausii, a supposed alkalophilic ortholog of cotA from B. subtilis. Both laccases were expressed in E. coli strain BL21(DE3) and characterized fully in parallel for strict benchmarking. We report activity on ABTS, SGZ, DMP, caffeic acid, promazine, phenyl hydrazine, tannic acid, and bilirubin at variable pH. Whereas ABTS, promazine, and phenyl hydrazine activities vs. pH were similar, the activity of B. clausii cotA was shifted upwards by ~0.5-2 pH units for the simple phenolic substrates DMP, SGZ, and caffeic acid. This shift is not due to substrate affinity (K(M)) but to pH dependence of catalytic turnover: The k(cat) of B. clausii cotA was 1 s⁻¹ at pH 6 and 5 s⁻¹ at pH 8 in contrast to 6 s⁻¹ at pH 6 and 2 s⁻¹ at pH 8 for of B. subtilis cotA. Overall, k(cat)/K(M) was 10-fold higher for B. subtilis cotA at pH(opt). While both proteins were heat activated, activation increased with pH and was larger in cotA from B. clausii. NaCl inhibited activity at acidic pH, but not up to 500-700 mM NaCl in alkaline pH, a further advantage of the alkali regime in laccase applications. The B. clausii cotA had ~20 minutes half-life at 80°C, less than the ~50 minutes at 80°C for cotA from B. subtilis. While cotA from B. subtilis had optimal stability at pH~8, the cotA from B. clausii displayed higher combined salt- and alkali-resistance. This resistance is possibly caused by two substitutions (S427Q and V110E) that could repel anions to reduce anion-copper interactions at the expense of catalytic proficiency, a trade-off of potential relevance to laccase optimization

Structural and functional analysis of the gpsA gene product of Archaeoglobus fulgidus: A glycerol-3-phosphate dehydrogenase with an unusual NADP(+) preference

NAD(+)-dependent glycerol-3-phosphate dehydrogenase (G3PDH) is generally absent in archaea, because archaea, unlike eukaryotes and eubacteria, utilize glycerol-1-phosphate instead of glycerol-3-phosphate for the biosynthesis of membrane lipids. Surprisingly, the genome of the hyperthermophilic archaeon Archaeoglobus fulgidus comprises a G3PDH ortholog, gpsA, most likely due to horizontal gene transfer from a eubacterial organism. Biochemical characterization proved G3PDH-like activity of the recombinant gpsA gene product. However, unlike other G3PDHs, the up to 85°C thermostable A. fulgidus G3PDH exerted a 15-fold preference for NADPH over NADH. The A. fulgidus G3PDH bears the hallmarks of adaptation to halotolerance and thermophilicity, because its 1.7-Å crystal structure showed a high surface density for negative charges and 10 additional intramolecular salt bridges compared to a mesophilic G3PDH structure. Whereas all amino acid residues required for dihydroxyacetone phosphate binding and reductive catalysis are highly conserved, the binding site for the adenine moiety of the NAD(P) cosubstrate shows a structural variation that reflects the observed NADPH preference, for example, by a putative salt bridge between R49 and the 2′-phosphate

Precipitation of carbides in F82H steels and its impact on mechanical strength

The precipitation of carbides in F82H steel and its model steel (Fe-0.2TaC) were investigated by transmission electron microscopy (TEM) and extracted residue tests (ERT). The effects of tempering on the precipitation in F82H steels were elucidated on the basis of the characterization of carbides, as well as the quantitative estimation of precipitation strengthening at room temperature. Firstly, the number density of precipitates was measured by extracted residue test in Fe-0.2TaC and tempered F82H steel, and compared with the TEM observation. It was found that the ratio of volume fraction between the TEM and the ERT was respectively 1.84 and 0.54 for Fe-0.2TaC and tempered F82H steel, revealing that the collection probability of ERT strongly depends on the precipitate features, size and number density. Effects of annealing on the precipitation in F82H steels were investigated by ERT. The amount of carbide showed a non-linear relationship to tempering parameter, TP  =  T(20 + logt). It steeply increased in the TP range from 15.3 to 16.2. The precipitation strengthening in F82H steel was estimated to be about 5–10% relative to its proof strength, suggesting that the carbides in F82H steel have a minor role on the tensile strength at room temperature, though these precipitates are greatly beneficial for improving the creep and radiation resistance at elevated temperatures

Microstructure and mechanical property in heat affected zone (HAZ) in F82H jointed with SUS316L by fiber laser welding

This study investigates the microstructure and mechanical property in heat affected zone (HAZ) between F82H and SUS316L jointed by 4 kW fiber laser welding at different parameters such as laser scan rate and beam position. OM/FE-SEM observation, EPMA analysis and nano-indentation hardness test were utilized to characterize the microstructure and evaluate the mechanical property. Results show that the HAZ width is dependent on the welding condition. The precipitation of M23C6 particle in HAZ is found to be closely related to the distance from WM/HAZ interface. Decrease in Cr and C concentration in M23C6 depended on the welding condition; the decrease was relatively milder in the case of shifting the beam position to SUS side. Furthermore, the rapid increment in nano-indentation hardness, i.e. ≈2500 MPa, at HAZ/F82H interface was observed regardless of welding parameters. The temperatures at HAZ/F82H interface were estimated from Cr and C concentration change of M23C6 by EPMA. It was revealed that the temperature of HAZ/F82H interface increased with increasing HAZ width, and that the presence of over-tempered HAZ (THAZ) region is confirmed only in the specimens welded right on the F82H/SUS interface (no-shift) at the laser scan rate of 3 m/min

Experimental Assessment of Temperature Distribution in Heat Affected Zone (HAZ) in Dissimilar Joint between 8Cr-2W Steel and SUS316L Fabricated by 4 kW Fiber Laser Welding

High-Cr steel (Fe-8Cr-2W-0.4Mn-0.2V-0.06Ta, so-called F82H steel) is one of the candidate structure materials for fusion blanket system, and its dissimilar welding technology to stainless steel (SUS316L) is an inevitable issue. Fiber laser welding is a promising technique to joint F82H and SUS materials, because of its non-mandatory vacuum environment during welding, and the reduced distortion and residual stress. Purpose of the present study is to identify the microstructure characteristics of dissimilar joint between F82H and SUS316L fabricated by fiber laser welding. M23C6-type precipitates were evaluated using an electron back-scatter diffraction device equipped with a scanning electron microscope, an electron probe micro analyzer, and a transmission electron microscope. The microstructure of as-welded specimens was composed of heat affected zone (coarse grain (CGHAZ) and fine grain (FGHAZ)) in F82H side, and weld metal (WM). Distributions of size, density and chemical composition of precipitates in HAZ were examined as a function of the distance from WM/HAZ interface. Besides, isochronal annealing experiments on F82H performed to obtain relationship between annealing temperature and Cr (or C) concentration in M23C6 precipitates. Cr-rich M23C6 comparing with original F82H (tempered F82H) one was observed at temperature ranging from 1103 to 1173 K. By comparison of their microstructure with that of HAZ, temperature distribution in HAZ upon welding was evaluated. It is suggested that the HAZ/F82H interface and the FGHAZ/CGHAZ boundary were heated up to 1150 K and 1400 K, respectively. Further applications of this method to clarify the temperature history and its distribution in HAZ, as well as increase in diagnostic accuracy, are greatly expected