Improved expression and characterization of recombinant human Golgi α1,2-mannosidase I isoforms (IA2 and IC) by Escherichia coli

Golgi α1,2-mannosidase I is involved in the N-linked oligosaccharide processing pathway. In this study, two truncated genes encoding for human Golgi α1,2-mannosidase I (hManIA2: amino acids 127-626 and hManIC: amino acids 118-617) were expressed in Escherichia coli to characterize the enzymes. These genes were fused to a T7 protein tag and a histidine tag at the N- and C-terminal ends, respectively, and purified using Co(2+) affinity chromatography. The properties including optimal temperature, optimal pH, and substrate specificity of the purified enzymes were investigated by HPLC using pyridylamino (PA)-labeled oligosaccharides as substrates. The stability of hManIA2 was dependent on the presence of Ca(2+), which was also required for its activity. On the other hand, hManIC was stable in the absence of Ca(2+), even though Ca(2+) was also effective for the activity of hManIC. While the similarity of the amino acid sequences is over 60%, hManIA2 and hManIC showed different substrate specificities particularly toward M9A and M8C.     

Characteristics of enterotoxin H-producing Staphylococcus aureus isolated from clinical cases and properties of the enterotoxin productivity

Staphylococcal enterotoxin H (SEH) is predicted to be involved in staphylococcal food poisoning. To characterize SEH-producing Staphylococcus aureus isolates from staphylococcal food poisoning cases in Japan, we investigated the relationship between SEH production and coagulase serotype, which is an epidemiological marker, and compared the properties of SEH production with those of staphylococcal enterotoxins A (SEA) and B (SEB). SEH production was determined by a newly developed sandwich enzyme-linked immunosorbent assay. Eighty-six (59.7%) of 144 isolates from staphylococcal food poisoning cases produced SEH. Seventy-one of the SEH-producing isolates simultaneously produced SEA, SEB, or both. All SEH-producing isolates belonged to coagulase type VII, which was the predominant type, representing 99 (68.8%) of 144 isolates. The amount of SEH produced in brain heart infusion was almost the same as the amount of SEA and approximately 10-fold lower than that of SEB. SEH and SEA were produced mainly during the late exponential phase of growth, whereas SEB was produced mostly during the stationary phase. The production levels of SEH and SEA were gradually affected by decreases in water activity, but the production of SEB was greatly reduced under conditions of low water activity. These findings indicate that SEH-producing S. aureus isolates are of high prevalence in staphylococcal food poisoning cases. Given the unique epidemiological characteristic of these isolates, SEH and SEA probably are responsible for food poisoning.     

Model-based reconstruction integrated with fluence compensation for photoacoustic tomography

Photoacoustic (PA) tomography (PAT) is a rapidly developing imaging modality that can provide high contrast and spatial-resolution images of light-absorption distribution in tissue. However, reconstruction of the absorption distribution is affected by nonuniform light fluence. This paper introduces a reconstruction method for reducing amplification of noise and artifacts in low-fluence regions. In this method, fluence compensation is integrated into model-based reconstruction, and the absorption distribution is iteratively updated. At each iteration, we calculate the residual between detected PA signals and the signals computed by a forward model using the initial pressure, which is the product of estimated voxel value and light fluence. By minimizing the residual, the reconstructed values converge to the true absorption distribution. In addition, we developed a matrix compression method for reducing memory requirements and accelerating reconstruction speed. The results of simulation and phantom experiments indicate that the proposed method provides a better contrast-to-noise ratio (CNR) in low-fluence regions. We expect that the capability of increasing imaging depth will broaden the clinical applications of PAT.     

Continuous synthesis of glyceryl ferulate using immobilized Candida antarctica lipase

Glyceryl ferulate was synthesized through condensation of ferulic acid and glycerol at 50 degrees C in glycerols with different water contents using an immobilized lipase from Candida antarctica in a batch reactor, and condensation in the glycerol with a 7.5% (w/w) water content was shown to be the favorite. The solubility of ferulic acid was higher at higher temperature in glycerol with a lower water content. The viscosity was lower at higher temperature for the glycerol with a higher water content. The condensation was carried out using a batch reactor at a temperature from 50 degrees C to 90 degrees C. These observations indicated that the condensation at 80 degrees C in the glycerol with a 7.5% (w/w) water content was the most adequate for continuously synthesizing glyceryl ferulate. A reactor system was constructed for the continuous synthesis and was steadily operated to realize a productivity of 430 kg/(m(3)-reactor(day) without any decrease in the conversion for at least 6 days.     

Purification of Arachidonic acid from <Emphasis Type="Italic">Mortierella</Emphasis> single-cell oil by selective esterification with <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> lipase

Purification of arachidonic acid (AA) from Mortierella alpina single-cell oil was attempted. The process comprised three steps: (i) preparation of FFA by nonselective hydrolysis of the oil with Alcaligenes sp. lipase; (ii) elimination of long-chain saturated FA from the resulting FFA by urea adduct fractionation; and (iii) enrichment of AA through lipase-catalyzed selective esterification with lauryl alcohol (LauOH). In the third step, screening of industrially available lipases indicated that Burkholderia cepacia lipase (Lipase-PS, Amano Enzyme Inc., Aichi, Japan) acted on AA more weakly than on other FA and was the most effective for enrichment of AA in the FFA fraction. When the FFA obtained by urea adduct fractionation were esterified with 2 molar equivalents of LauOH at 30°C for 16 h in a mixture with 20% water and 20 units (U)/g-mixture of Lipase-PS, the esterification reached 39% and the content of AA in the FFA fraction was raised from 61 to 86 wt%. To further increase the content of AA, unesterified FFA were allowed to react again under the same conditions as those in the first selective esterification except for the use of 50 U/g Lipase-PS. A series of procedures raised the content of AA to 97 wt% with a 49% recovery based on the initial content in the single-cell oil. These results indicated that the three-step process for selective esterification with Lipase-PS was effective for purifying AA from the single-cell oil.     

Melt fracture behavior of polypropylene‐type resins with narrow molecular weight distribution. II. Suppression of sharkskin by addition of adhesive resins

Standing on a hypothesis that the sharkskin of a polymer with a narrow molecular weight distribution at extrusion processing originates from a stick‐slip of the polymer at the die wall, the suppression of the sharkskin was tried by means of suppressing the slip by the addition of adhesives. To polypropylene (PP)‐type resins with narrow molecular weight distributions such as a PP‐type thermoplastic elastomer, PER and a controlled rheology PP were added small amounts of adhesives such as maleated PP, maleated PER, reactive polyolefin oligomers, ethylene/ethylacrylate/maleic anhydride (MAH) copolymer, ethylene/vinyl acetate copolymer, and styrene/MAH copolymer, and their melt fracture behaviors at capillary extrusion were observed. It was found that the sharkskin of the PP‐type resins with narrow molecular weight distributions was suppressed by the addition of the adhesive resins with good adhesion to metal. The suppressive effect of the sharkskin was generally the more remarkable by the higher loading of the adhesives with the higher MAH content. This is the direction of increasing adhesion. From this fact, it was assumed that the sharkskin of the PP‐type resins with narrow molecular weight distribution does not originate from a periodic growth and relaxation of tensile stress at the extrudate surface but from a stick‐slip at the die wall. Based on this mechanism, it may be said that the sharkskin can be suppressed by both ways of directions of promoting and suppressing the slip at the die wall. The former way is the previously known method, and the latter way is the method proposed in the present study. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 84: 2120–2127, 2002     

RHO gene products, putative small GTP-binding proteins, are important for activation of the CAL1/CDC43 gene product, a protein geranylgeranyltransferase in Saccharomyces cerevisiae.

Two multicopy suppressors of the cal1-1 mutation in the yeast Saccharomyces cerevisiae have been isolated and characterized. They are identical to the yeast RHO1 and RHO2 genes, which encode putative small GTP-binding proteins. Multiple copies of either RHO gene suppressed temperature-sensitive growth of the cal1-1 mutant but did not suppress the cal1 null mutant. Genetic analysis suggests that overproduction of either RHO gene product acts for activation of the CAL1 gene product.     

A technique of parallel connections of pulsewidth-modulated NPC inverters by using current-sharing reactors

The neutral-point-clamped (NPC) PWM inverters have many advantages such as the ability to operate the motor with nearly sinusoidal current waveforms. For this reason, in larger-capacity inverter systems, NPC PWM inverters using the GTOs, etc., have also been put into practical use because of great advantages to large-capacity ac motor drives such as lower ripple currents and higher output voltages. With the spread of applications, still larger-capacity inverters also are expected. However, since the capacities of such switching devices are insufficient, a certain technique and controlling method are proposed for the parallel connections of NPC inverters. The output voltage waveforms of the proposed inverter have certain voltage levels, and thus it is anticipated that it will be difficult to analyze the output harmonics. For such waveforms of output voltage and current-sharing reactor, a frequency analysis approach is described whose results are verified by experiments.     

Creep damage assessment of 10Cr-1Mo-1W-VNbN steel forging through EBSD observation

10Cr-1Mo-1W-VNbN steel forging was observed through TEM (Transmission Electron Microscope), SEM (Scanning Electron Microscope) with EBSD (Electron BackScattering Diffraction) pattern method and tested through nano-indentation tester to investigate the microstructural change during creep damage process. Long-term creep rupture and interrupted creep test samples were investigated and effective damage parameters were selected. Dislocation substructure through TEM thin foil method showed increasing block/lath width especially near grain boundary according to creep damage accumulation and the same feature was observed through EBSD IPF mapping more clearly and easily. Area averaged KAM (Kernal Average Misorientation) KAM_ave was shown to be effective for evaluating dislocation microstructural changes during creep. Nano-indentation tests were conducted at the same position in EBSD measurement, which showed a good correlation between hardness value and the square root of KAM_ave. The differential equation of dislocation density with creep time was applied to estimate the relationship between averaged KAM_ave and time through the relationship between hardness and dislocation density. The creep damage estimation curves were obtained by the integrated form of the equation. As the KAM_ave showed an apparent drop against time fraction in the primary creep stage near grain boundary followed by almost constant trend for later stage. The statistical distribution of KAM_ave during creep damage process suggested the localized recovery of dislocation substructure near grain boundary.