Uniform culture in solid-state fermentation with fungi and its efficient enzyme production

Solid-state fermentation (SSF) has attracted a lot of interest for carrying out high-level protein production in filamentous fungi. However, it has problems such as the fermentation heat generated during the culture in addition to the reduced mobility of substances. These conditions lead to a nonuniform state in the culture substrate and result in low reproducibility. We constructed a non-airflow box (NAB) with a moisture permeable fluoropolymer membrane, thereby making it possible to control and maintain uniform and optimal conditions in the substrate. For the NAB culture in Aspergillus oryzae, temperature and water content on/in the whole substrate were more consistent than for a traditional tray box (TB) culture. Total weight after the culture remained constant and dry conditions could be achieved during the culture. These data demonstrate the possibility of growing a uniform culture of the whole substrate for SSF. The NAB is advantageous because it allows for the control of exact temperature and water content in the substrate during the culture by allowing vapor with latent heat to dissipate out of the box. In addition, several enzymes in the NAB culture exhibited higher production levels than in the TB culture. We believe that culturing in the constructed NAB could become a standard technique for commercial SSF.     

Ironmaking with ammonia at low temperature

This paper describes the reduction of hematite with ammonia for ironmaking, in which the effect of temperature on the products was examined. The results showed that the reduction process began at 430 °C during heating, and with an increase in temperature, the reduction mechanism changed apparently from a direct reduction of ammonia (Fe(2)O(3) + 2NH(3) → 2Fe + N(2) + 3H(2)O) to an indirect reduction via the thermal decomposition of ammonia (2NH(3) → N(2) + 3H(2), Fe(2)O(3) + 3H(2) → 2Fe + 3H(2)O) at temperatures over 530 °C. The final product obtained at 600 and 700 °C was pure metallic iron, in contrast with that formed at 450 °C, that is, a mixture of metallic iron and iron nitride. The results suggest the possibility of using ammonia as a reducing agent for carbonless ironmaking, which is operated at a much lower temperature than 900 °C in conventional coal-based ironmaking.     

Steric hindrance by 2 amino acid residues determines the substrate specificity of isomaltase from Saccharomyces cerevisiae

The structures of the E277A isomaltase mutant from Saccharomyces cerevisiae in complex with isomaltose or maltose were determined at resolutions of 1.80 and 1.40 Å, respectively. The root mean square deviations between the corresponding main-chain atoms of free isomaltase and the E277Α-isomaltose complex structures and those of free isomaltase and the E277A-maltose complex structures were found to be 0.131 Å and 0.083 Å, respectively. Thus, the amino acid substitution and ligand binding do not affect the overall structure of isomaltase. In the E277A-isomaltose structure, the bound isomaltose was readily identified by electron densities in the active site pocket; however, the reducing end of maltose was not observed in the E277A-maltose structure. The superposition of maltose onto the E277A-maltose structure revealed that the reducing end of maltose cannot bind to the subsite + 1 due to the steric hindrance from Val216 and Gln279. The amino acid sequence comparisons with α-glucosidases showed that a bulky hydrophobic amino acid residue is conserved at the position of Val216 in α-1,6-glucosidic linkage hydrolyzing enzymes. Similarly, a bulky amino acid residue is conserved at the position of Gln279 in α-1,6-glucosidic linkage-only hydrolyzing α-glucosidases. Ala, Gly, or Asn residues were located at the position of α-1,4-glucosidic linkage hydrolyzing α-glucosidases. Two isomaltase mutant enzymes – V216T and Q279A – hydrolyzed maltose. Thus, the amino acid residues at these positions may be largely responsible for determining the substrate specificity of α-glucosidases.     

Preoperative staging of endometrial carcinoma: diagnostic effect of T2-weighted fast spin-echo MR imaging

PURPOSE: To evaluate the usefulness of T2-weighted fast spin-echo magnetic resonance (MR) imaging with a 512 x 256 matrix for assessment of the preoperative stage of endometrial carcinoma. MATERIALS AND METHODS: Twenty-eight women with histopathologically proved endometrial carcinoma underwent preoperative T2-weighted fast spin-echo, dynamic T1-weighted fast spin-echo, and postcontrast T1-weighted spin-echo MR imaging with a phased-array surface coil. The uterine long-axis planes in each sequence were reviewed at separate sessions by three radiologists blinded to the histopathologic data. RESULTS: For the diagnosis of myometrial invasion, no statistically significant differences were found among T2-weighted imaging, dynamic imaging, and postcontrast T1-weighted imaging. For the diagnosis of deep myometrial invasion, T2-weighted and dynamic images showed higher specificity than postcontrast T1-weighted images (T2-weighted, 89%; dynamic, 88%; and postcontrast T1-weighted, 80%). For cervical invasion, T2-weighted and dynamic images showed larger areas under receiver operating characteristic curves than did postcontrast T1-weighted images (T2-weighted, 0.78; dynamic, 0.71; and postcontrast T1-weighted, 0.67). CONCLUSION: T2-weighted imaging is useful for identifying the stage of endometrial carcinoma.     

Enhancement of BTI degradation in pMOSFETs under high-frequency bipolar gate bias

Negative bias temperature (NBT) instability of p-MOSFETs with ultrathin SiON gate dielectric has been investigated under various gate bias configurations. The NBT-induced interface trap density (/spl Delta/N/sub it/) under unipolar bias is essentially lower than that under static bias, and is almost independent of the stress frequency up to 10 MHz. On the contrary, /spl Delta/N/sub it/ under bipolar pulsed bias of frequency larger than about 10 kHz is significantly enhanced and exhibits a strong frequency dependence, which has faster generation rate and smaller activation energy as compared to other stress configurations. The degradation enhancement is attributed to the energy to be contributed by the recombination of trapped electrons and free holes upon the silicon surface potential reversal from accumulation to inversion.     

Heat Transfer & Thermal Stress Analysis of Plastic-Encapsulated ICs

Analytic approaches of thermal stress in plastic-encapslated ICs reliability have been studied using a simple, 2-dimensional model of the cross section of ICs by the finite-element method. To test the validity of the model, the actual stress within the silicon chip was measured using piezoresistive devices. The calculated stress in the silicon chip agreed with the experimental values. The stress distributions were changed by lead-frame properties. Package cracking, and delamination between the molding plastic and the lead-frame were qualitatively explained. We estimated the effect of the plastic properties on stress quantitatively. Furthermore, to test the validity of this model, the temperature change at the silicon chip was measured using the Vf temperature dependency of a diode. The calculated temperature change at the silicon chip agreed with the observed values. A very high temperature gradient was observed near the surface of the plastic immediately after solder dipping. The non-uniform temperature distributions produced different thermal stress distributions than those observed in the steady-state. This result indicated that rapid thermal shock could delaminate the plastic from the lead-frame. We believe that these results can guide the development of an optimum low-stress plastic.     

Numerical analysis of bubble motion with the VOF method

Numerical analyses of a two-dimensional single bubble in a stagnant liquid and in a linear shear flow were conducted in the present study using the volume of fluid method, which is based on the local-instantaneous field equations. It was clarified that this method gives qualitatively appropriate predictions for the effects of the Morton number and the Eotvos number on fluctuating bubble motion in a stagnant liquid. Calculated velocity and pressure distributions indicated that the Karman vortex causes a sinuous movement of the bubble. As for the bubble motion in a linear shear flow, calculated bubbles migrated in a lateral direction. The direction of the lateral migration agreed to available experimental data. It was also confirmed that (i) the direction or the magnitude of the lateral migration is affected by the Eotvos and the Morton numbers, and (ii) the interaction among the internal flow of the bubble, the wake of the bubble and the external shear flow plays an essential role for the lateral migration.     

Increase in adhesion molecules on CD4+ cells and CD4+ cell subsets in synovial fluid from patients with rheumatoid arthritis

OBJECTIVE: To elucidate the role of adhesion molecules in the pathogenesis of rheumatoid arthritis (RA). METHODS: We evaluated their expression and that of an activation marker on CD4+ cell populations and CD4+ cell subsets in specimens of peripheral blood (PB) and synovial fluid (SF) obtained from 10 patients with RA and 7 with osteoarthritis (OA). A 2 or 3-color immunofluorescent method was used for analysis. RESULTS: The SF from both groups of patients showed a greater density of adhesion molecules including LFA-1 alpha, LFA-1 beta, CD2, VLA-4 alpha and VLA-5 alpha on CD4+ cells, and a higher percentage of CD4+HLA-DR+ cells compared with their PB. IN PB-CD4+ cell subsets from the arthritic and healthy subjects, the CD4+CD45RO+ cell population showed an increased expression of adhesion molecules compared with CD4+CD45RA+ cell population. The expression of adhesion molecules on circulating CD4+ cell population and CD4+ cell subsets from the patients with RA and OA was comparable to that from healthy subjects. SF from both groups of patients showed a higher percentage of CD4+CD45RO+ cells and a lower percentage of CD4+CD45RA+ cells. In SF-CD4+ cell subsets from patients with RA, the CD4+CD45RO+ cell population had an increased expression of VLA-4 alpha compared to the CD4+CD45RA+ cell population; however, there was no significant difference in other adhesion molecule expression and the percentage of HLA-DR+ cells between the 2 cell subsets. Furthermore, the expression of VLA-4 alpha on the CD4+CD45RO+ cell population in SF from patients with RA was significantly higher than that in matched PB. In CD4+CD45RA+ cell population from both groups of patients, SF showed an enhanced expression of adhesion molecules and an increased percentage of HLA-DR+ cells compared with matched PB. CONCLUSION: Our results suggest that increased expression of adhesion molecules and increased percentage of HLA-DR+ cells on CD4+ cells in SF may be responsible for cellular interactions between these cells and synovial cells or extracellular matrix.     

Clonal heterogeneity in human esophageal squamous cell carcinomas on DNA analysis

Cancers are thought to arise through multistep accumulation of somatic mutations in the progeny of a single cell. Multiple mutations may induce molecular intratumor heterogeneity. Therefore, we examined molecular clonal heterogeneity in esophageal squamous cell carcinomas. Twenty-four esophageal squamous cell carcinomas and associated lymph node metastases were examined for microsatellite alterations, and abnormalities of the p53 and transforming growth factor-beta type II receptor (TGF-beta RII) genes. There were eight cases (33%) showing different patterns of loss of heterozygosity in primary tumors and metastatic lymph nodes with microsatellite markers. On the other hand, the abnormalities of p53 were identical in all these cases. No mutation was detected in the simple repeated sequences of the TGF-beta RII gene. These results indicate that molecular clonal heterogeneity exists in esophageal squamous cell carcinomas. Therefore, care is necessary in preoperative genetic diagnosis using biopsy samples.