Preparation of Mutants Resistant to Catabolite Repression of Trichoderma reesei

The development of agar plate screening techniques has allowed the isolation of mutants of Trichoderma reesei capable of synthesizing cellulase under the conditions of a high concentration of glucose. Mutants resistant to catabolite repression by glycerol or glucose were isolated on Walseth’s cellulose (WC) agar plates containing 5% glycerol or 5% glucose, respectively. Mutants resistant to catabolite repression by glycerol were not derepressed enough for the production of cellulase on WC agar plates containing 5% glucose or in flask cultures with a mixture of 1% Avicel and 3% glucose. On the contrary, two mutant strains resistant to catabolite repression by glucose (KDD-10 and DGD-16) produced large clearing zones on WC agar plates containing 5% glucose. Both strains could begin to produce CMCase even in the presence of residual glucose and finally produced 1.5 times the CMCase activity, in flask cultures on 1% Avicel and 3% glucose, than that with 1% Avicel alone. These results suggest that KDD-10 and DGD-16 are comparatively derepressed by glucose for cellulase production.     

Nicotine Dependence and Cost-Effectiveness of Individualized Support for Smoking Cessation: Evidence from Practice at a Worksite in Japan

Given the lack of economic studies evaluating the outcomes of smoking cessation programs from the viewpoint of program sponsors, we conducted a case study to provide relevant information for worksites. The present study was carried out between 2006 and 2008 at a manufacturing factory in the Toyama Prefecture of Japan and included subjects who voluntarily entered a smoking cessation program. The program included face-to-face counselling followed by weekly contact to provide encouragement over six months using e-mail or inter-office mail. Nicotine patches were available if required. All 151 participants stopped smoking immediately. Over the 24-month study period, self-report showed 49.7% abstained continuously from smoking. The rate of 24-month consecutive abstinence was higher in participants with lower Fagerström Test scores for Nicotine Dependence at baseline than in those with higher scores (63.6% for 0–2 points vs. 46.5% for 3–6 points vs. 43.8% for 7–10 points; chi-square test p = 0.19). A logistic regression model showed a significant linear trend for the association between the score and abstinence status after adjustment for possible confounding factors (p = 0.03). The crude incremental cost for one individual to successfully quit smoking due to the support program was ¥46,379 (i.e., ¥100 = $1.28, £0.83, or €1.03 at foreign exchange rates). The corresponding costs for the three categories of the Fagerström Test score for Nicotine Dependence were ¥31,953, ¥47,450 and ¥64,956, respectively. When a sensitivity analysis was conducted based on the 95% confidence interval of the success rate, the variance in the corresponding costs was ¥25,514–45,034 for 0–2 points, ¥38,344–61,824 for 3–6 points, and ¥45,698–108,260 for 7–10 points. The degree of nicotine dependence may therefore be an important determinant of the cost-effectiveness of smoking cessation programs.     

Structural Study of Cell Attachment Peptide Derived from Laminin by Molecular Dynamics Simulation

Peptides with cell attachment activity are beneficial component of biomaterials for tissue engineering. Conformational structure is one of the important factors for the biological activities. The EF1 peptide (DYATLQLQEGRLHFMFDLG) derived from laminin promotes cell spreading and cell attachment activity mediated by α2β1 integrin. Although the sequence of the EF2 peptide (DFATVQLRNGFPYFSYDLG) is homologous sequence to that of EF1, EF2 does not promote cell attachment activity. To determine whether there are structural differences between EF1 and EF2, we performed replica exchange molecular dynamics (REMD) simulations and conventional molecular dynamics (MD) simulations. We found that EF1 and EF2 had β-sheet structure as a secondary structure around the global minimum. However, EF2 had variety of structures around the global minimum compared with EF1 and has easily escaped from the bottom of free energy. The structural fluctuation of the EF1 is smaller than that of the EF2. The structural variation of EF2 is related to these differences in the structural fluctuation and the number of the hydrogen bonds (H-bonds). From the analysis of H-bonds in the β-sheet, the number of H-bonds in EF1 is larger than that in EF2 in the time scale of the conventional MD simulation, suggesting that the formation of H-bonds is related to the differences in the structural fluctuation between EF1 and EF2. From the analysis of other non-covalent interactions in the amino acid sequences of EF1 and EF2, EF1 has three pairs of residues with hydrophobic interaction, and EF2 has two pairs. These results indicate that several non-covalent interactions are important for structural stabilization. Consequently, the structure of EF1 is stabilized by H-bonds and pairs of hydrophobic amino acids in the terminals. Hence, we propose that non-covalent interactions around N-terminal and C-terminal of the peptides are crucial for maintaining the β-sheet structure of the peptides.     

Novel dual-reporter transgenic rodents enable cell tracking in animal models of stem cell transplantation

In the present study, we have established a novel transgenic mouse and transgenic rats with dual reporters of EGFP and ELuc. In these transgenic (Tg) rodents, both GFP fluorescent and luciferase luminescent signals were ubiquitously detected in the heart, liver, kidney and testis, while only the GFP signal was detected in the brain. This expression system is based on a P2A linked EGFP/ELuc protein allowing both signals to be generated simultaneously. Microscopy experiments, FCM, and luciferase assays showed strong expression in freshly isolated ADSCs from Tg rodents upon transplantation of Tg rat-derived ADSCs into wild-type-mice. The ELuc transgene signal was observed and traced in vivo, and EGFP positive cells could be recovered from ELuc positive tissues in engraftment sites of wild-type mice for multiple analysis. These dual reporter Tg rodents are a useful reconstituted model system of regenerative medicine and are a valuable tool to study stem cells.     

Do mammalian cells synthesize lipoic acid? Identification of a mouse cDNA encoding a lipoic acid synthase located in mitochondria

Lipoic acid is a coenzyme essential to the activity of enzymes such as pyruvate dehydrogenase, which play important roles in central metabolism. However, neither the enzymes responsible for biosynthesis nor the biosynthetic event of lipoic acid has been reported in mammalian cells. In this study, a mouse mLIP1 cDNA for lipoic acid synthase has been identified. We have shown that the cDNA encodes a lipoic acid synthase by its ability to complement a mutant of Escherichia coli defective in lipoic acid synthase and that mLIP1 is targeted into the mitochondria. These findings suggest that mammalian cells are able to synthesize lipoic acid in mitochondria.     

Optimization of agitation and aeration conditions for maximum virginiamycin production

To maximize the productivity of virginiamycin, which is a commercially important antibiotic as an animal feed additive, an empirical approach was employed in the batch culture of Streptomyces virginiae. Here, the effects of dissolved oxygen (DO) concentration and agitation speed on the maximum cell concentration at the production phase, as well as on the productivity of virginiamycin, were investigated. To maintain the DO concentration in the fermentor at a certain level, either the agitation speed or the inlet oxygen concentration of the supply gas was manipulated. It was found that increasing the agitation speed had a positive effect on the antibiotic productivity independent of the DO concentration. The optimum DO concentration, agitation speed and addition of an autoregulator, virginiae butanolide C (VB-C), were determined to maximize virginiamycin productivity. The optimal strategy was to start the cultivation at 450 rpm and to continue until the DO concentration reached 80%. After reaching 80%, the DO concentration was maintained at this level by changing the agitation speed, up to a maximum of 800 rpm. The addition of an optimal amount of the autoregulator VB-C in an experiment resulted in the maximal production of virginiamycin M (399 mg/l), which was about 1.8-fold those obtained previously. Received: 13 July 1998 / Received revision: 19 August 1998 / Accepted: 13 September 1998     

Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer

Fetal liver, the major site of hematopoiesis during embryonic development, acquires additional various metabolic functions near birth. Although liver development has been characterized biologically as consisting of several distinct steps, the molecular events accompanying this process are just beginning to be characterized. In this study, we have established a novel culture system of fetal murine hepatocytes and investigated factors required for development of hepatocytes. We found that oncostatin M (OSM), an interleukin-6 family cytokine, in combination with glucocorticoid, induced maturation of hepatocytes as evidenced by morphological changes that closely resemble more differentiated hepatocytes, expression of hepatic differentiation markers and intracellular glycogen accumulation. Consistent with these in vitro observations, livers from mice deficient for gp130, an OSM receptor subunit, display defects in maturation of hepatocytes. Interestingly, OSM is expressed in CD45(+) hematopoietic cells in the developing liver, whereas the OSM receptor is expressed predominantly in hepatocytes. These results suggest a paracrine mechanism of hepatogenesis; blood cells, transiently expanding in the fetal liver, produce OSM to promote development of hepatocytes in vivo.     

Gene cloning and biochemical characterizations of thermostable ribonuclease HIII from Bacillus stearothermophilus

The gene encoding RNase HIII from the thermophilic bacterium Bacillus stearothermophilus was cloned and overexpressed in Escherichia coli, and the recombinant protein (Bst-RNase HIII) was purified and biochemically characterized. Bst-RNase HIII is a monomeric protein with 310 amino acid residues, and shows an amino acid sequence identity of 47.1% with B. subtilis RNase HIII (Bsu-RNase HIII). The enzymatic properties of Bst-RNase HIII, such as pH optimum, metal ion requirement, and cleavage mode of the substrates, were similar to those of Bsu-RNase HIII. However, Bst-RNase HIII was more stable than Bsu-RNase HIII, and the temperature (T(1/2)) at which the enzyme loses half of its activity upon incubation for 10 min was 55 degrees C for Bst-RNase HIII and 35 degrees C for Bsu-RNase HIII. The optimum temperature for Bst-RNase HIII activity was also shifted upward by roughly 20 degrees C as compared to that of Bsu-RNase HIII. The availability of such a thermostable enzyme will facilitate structural studies of RNase HIII.