Improving protein secretion of a transglutaminase-secreting Corynebacterium glutamicum recombinant strain on the basis of 13C metabolic flux analysis

Corynebacterium glutamicum is known as a host species for amino acid production. This microorganism was recently noticed as a host that produces secreted proteins. In this study, we performed ¹³C metabolic flux analysis (¹³C-MFA) on a recombinant C. glutamicum strain that secretes a heterologous transglutaminase (TGase) to improve TGase secretion. For the ¹³C-MFA of a TGase-secreting C. glutamicum strain in batch cultivation, a ¹³C-labeling experiment and measurement of mass isotopomer distributions of proteinogenic amino acids were performed, and metabolic fluxes were determined considering the changes in fractional ¹³C-labeling of proteinogenic amino acids with respect to culture time. The TGase yield increased at the stationary phase but decreased toward its end. The results of ¹³C-MFA revealed that the flux from glycolysis to the TCA cycle gradually increased during TGase secretion. We speculate that the NADH/NAD⁺ ratio in the cells increases and that as a result, the specific glucose uptake rate decreases in the stationary phase because of the increased flux of the TCA cycle. Since it is expected that a decrease in the NADH/NAD⁺ ratio would improve the TGase yield, we tried to enhance lactate production in a TGase-secreting C. glutamicum strain to decrease cellular NADH levels by increasing the pH level in the culture. The TGase yield increased in 1.4-fold by increasing the pH from 6.7 to 7.2, indicating that the TGase yield was successfully improved on the basis of the ¹³C-MFA.     

High-Temperature Compressive Properties of TiC-Added Mo-Si-B Alloys

High-temperature compressive properties of two TiC-added Mo-Si-B alloys with nominal compositions of Mo-5Si-10B-7.5TiC (70Mo alloy) and Mo-6.7Si-13.3B-7.5TiC (65Mo alloy) (at.%) were investigated. The alloys were composed of four constituent phases: Mo solid solution (Mo_ss), Mo₅SiB₂, (Mo,Ti)C, and (Mo,Ti)₂C. The primary phases of the 70Mo and 65Mo alloys were Mo_ss and T₂, respectively. The compressive deformability of the 65Mo alloy was significantly limited even at 1600°C because of the elongated, coarse primary T₂ phase, whereas the 70Mo alloy had good compressive deformability and a high strength in the test-temperature range of 1000–1600°C; the peak stresses were 1800 MPa at 1000°C, 1230 MPa at 1200°C, and 350 MPa at 1600°C. At and above 1200°C, the peak stress values were more than double those of Mo-6.7Si-7.9B, Ti-Zr-Mo, and Mo-Hf-C alloys. The plastic strain in the 70Mo alloy at temperatures lower than the ductile–brittle transition temperature of T₂ was generated by plastic deformation of not only Mo_ss but also of (Mo,Ti)C and (Mo,Ti)₂C. This work indicates that (Mo,Ti)C and (Mo,Ti)₂C play an important role in determining the high-temperature strength and deformation properties of TiC-added Mo-Si-B alloys.     

Dictyostelium Differentiation-Inducing Factor-1 Promotes Glucose Uptake,at Least in Part,via an AMPK-Dependent Pathway in Mouse 3T3-L1 Cells

Differentiation-inducing factor-1 (DIF-1) is a chlorinated alkylphenone (a polyketide) found in the cellular slime mold Dictyostelium discoideum. DIF-1 and its derivative, DIF-1(3M) promote glucose consumption in vitro in mammalian cells and in vivo in diabetic rats; they are expected to be the leading antiobesity and antidiabetes compounds. In this study, we investigated the mechanisms underlying the actions of DIF-1 and DIF-1(3M). In isolated mouse liver mitochondria, these compounds at 2–20 μM promoted oxygen consumption in a dose-dependent manner, suggesting that they act as mitochondrial uncouplers, whereas CP-DIF-1 (another derivative of DIF-1) at 10–20 μM had no effect. In confluent mouse 3T3-L1 fibroblasts, DIF-1 and DIF-1(3M) but not CP-DIF-1 induced phosphorylation (and therefore activation) of AMP kinase (AMPK) and promoted glucose consumption and metabolism. The DIF-induced glucose consumption was reduced by compound C (an AMPK inhibitor) or AMPK knock down. These data suggest that DIF-1 and DIF-1(3M) promote glucose uptake, at least in part, via an AMPK-dependent pathway in 3T3-L1 cells, whereas cellular metabolome analysis revealed that DIF-1 and DIF-1(3M) may act differently at least in part.     

Mechanics of peristaltic locomotion and role of anchoring

Limbless crawling is a fundamental form of biological locomotion adopted by a wide variety of species, including the amoeba, earthworm and snake. An interesting question from a biomechanics perspective is how limbless crawlers control their flexible bodies in order to realize directional migration. In this paper, we discuss the simple but instructive problem of peristalsis-like locomotion driven by elongation–contraction waves that propagate along the body axis, a process frequently observed in slender species such as the earthworm. We show that the basic equation describing this type of locomotion is a linear, one-dimensional diffusion equation with a time–space-dependent diffusion coefficient and a source term, both of which express the biological action that drives the locomotion. A perturbation analysis of the equation reveals that adequate control of friction with the substrate on which locomotion occurs is indispensable in order to translate the internal motion (propagation of the elongation–contraction wave) into directional migration. Both the locomotion speed and its direction (relative to the wave propagation) can be changed by the control of friction. The biological relevance of this mechanism is discussed.     

Molecular characterization of the Tim9 homologue from the methylotrophic yeast Pichia methanolica

In this paper we describe molecular characterization of the TIM9 gene encoding the essential mitochondrial inner-membrane protein in the methylotrophic yeast Pichia methanolica. PmTIM9 contains two exons corresponding to a gene product of 89 amino acid residues and a 140 bp intron. The deduced amino acid sequence exhibited high identity to those of other yeast Tim9ps, and possessed two CX(3)C motifs that contained two cysteine residues conserved among small Tim family proteins. Moreover, PmTIM9 had the ability to partially suppress the temperature sensitivity of Saccharomyces cerevisiae strain tim9-3, suggesting that PmTIM9 is a functional homologue of the ScTIM9 gene.     

On-chip enzyme immunoassay of a cardiac marker using a microfluidic device combined with a portable surface plasmon resonance system

This paper reports a miniaturized immunosensor designed to determine a trace level cardiac marker, B-type natriuretic peptide (BNP), using a microfluidic device combined with a portable surface plasmon resonance (SPR) sensor system. Sample BNP solution was introduced into the microchannel after an immunoreaction with acetylcholine esterase-labeled antibody (conjugate), and only unbound conjugate was trapped on the BNP-immobilized surface in the flow channel. Then, the thiol compound generated by the enzymatic reaction with the trapped conjugate was accumulated on a gold thin film located downstream in the microchannel to monitor the real-time SPR angle shift. We achieved a detectable concentration range of 5 pg/mL-100 ng/mL by monitoring the SPR angle shift, which covers the required detection range for the BNP concentrations found in blood. This success resulted from the use of a T-shaped microfluidic device structure, which prevents the sample solution from flowing over the gold film used for SPR detection. We were able to measure trace levels of BNP peptide (15 fg) within 30 min since the procedure with our immunosensor is simpler than a multistep immunoassay through the simultaneous use of a labeled enzymatic reaction and the real-time monitoring of enzymatic product accumulation in the microfluidic device. We employed the procedure to detect serum BNP by using spiked samples in human serum and achieved satisfactory recovery for heat-treated samples to denature the esterase in the serum before the immunoreaction.     

Antioxidant properties of herbal extracts selected from screening for potent scavenging activity against superoxide anions

BACKGROUND: In a previous study with the electron spin resonance (ESR) spin‐trapping method on large‐scale screening with ethanol extracts of approximately 1000 kinds of herbs, four herbal extracts with prominently potent ability to scavenge superoxide anions were chosen, namely the extracts from Punica granatum (peel), Syzygium aromaticum (bud), Mangifera indica (kernel) and Phyllanthus emblica (fruit)). In the present study, these extracts were further examined to determine if they also scavenge hydroxyl radicals, by applying the ESR spin‐trapping method, and if they have heat resistance as a desirable characteristic feature. RESULTS: Experiments with the Fenton reaction and UV irradiation of hydrogen peroxide, both of which generate hydroxyl radicals, showed that all four extracts had potent ability to directly scavenge hydroxyl radicals. Each extract exerted more potent hydroxyl radical‐scavenging activity than mannitol and salicylic acid. Furthermore, the scavenging activities against superoxide anions and hydroxyl radicals of the extracts of P. granatum (peel), M. indica (kernel) and P. emblica (fruit) proved to be heat‐resistant. CONCLUSION: The four herbal extracts chosen from extensive screening possess desirable antioxidant properties. In particular, three of them are expected to be suitable for food processing in which thermal devices are used, because of their heat resistance. Copyright © 2008 Society of Chemical Industry     

Peroxisomal metabolism is regulated by an oxygen-recognition system through organelle crosstalk between the mitochondria and peroxisomes

In the present study using Pichia methanolica, it was found that expressions of methanol-metabolic enzymes were strictly regulated by the presence of oxygen, and that induction of alcohol oxidase (AOD) isozymes was completely dependent on oxygen concentrations. A proportion of AOD-isozyme species responded to oxygen conditions, e.g. in a low oxygen condition, Mod1p was dominant, but with an increase in the oxygen concentration, the ratio of Mod2p increased. The K(m) value of Mod1p for oxygen was ca. one-seventh lower than that of Mod2p (0.47 and 3.51 mM, respectively). This shows that Mod1p is suitable at low oxygen concentrations and Mod2p at high oxygen concentrations. Also, zymogram changes for AOD isozymes were observed by inhibition of respiratory chain activity. These indicated that P. methanolica has the ability to recognize oxygen conditions and the respiratory chain should participate in the sensor for available oxygen. These facts indicate that there is organelle crosstalk between mitochondria and peroxisomes through nucleus gene regulation in order to control the consumption balance of available oxygen between the mitochondrial respiratory chain and peroxisomal AODs.     

Electrochemical surface plasmon resonance measurement in a microliter volume flow cell for evaluating the affinity and catalytic activity of biomolecules

We developed an electrochemical surface plasmon resonance flow cell for the simultaneous measurement of the binding affinity and catalytic activity of bifunctional biomolecules. These measurements will be useful for evaluating the performance of such biomolecules as ribozyme and abzyme. The simultaneous measurements were performed on a gold surface modified with a multilayer consisting of poly-l-lysine and poly(styrene sulfonate) assembled with the layer-by-layer method using an enzyme-labeled monoclonal antibody as a model compound. We obtained the amount of immunocomplex formation from the surface plasmon resonance angle shift value by injecting the compound into the flow cell containing the multilayer modified with tumor necrosis factor-alpha. Then we compared this surface plasmon resonance result with that for the in situ electrochemical oxidation of p-aminophenol formed by the catalytic reaction of labeled enzyme on the same gold film. We were able to obtain a high correlation coefficient of 0.999 between the two responses. This is because the compound could be captured with high stability with a less than 3% coulometric response decrease in the catalyzed product in the multilayer whose thickness was easily controllable. In addition, we were able to measure the catalytic activity by coulometry and thus avoid the effect of peak broadening. We also report that the dephosphorylation activity of a bound compound could be estimated from the measurement results and an equation.     

Systematic mapping of autonomously replicating sequences on chromosome V of Saccharomyces cerevisiae using a novel strategy

We have developed a new procedure for easy and rapid identification of autonomously replicating sequences (ARSs) and have applied it to the analysis of chromosome V of Saccharomyces cerevisiae. The procedure makes use of the ordered λ phage clone bank of this chromosome that we have constructed, and includes transposition of a mini-transposon and selection of transposon-containing derivatives, isolation of their DNA and circularization at their cos-ends, transformation of yeast cells with the circularized DNA, and scoring transformation frequency. The transposon used was derived from Tn5supF, contained the yeast LEU2 gene, and was placed, together with the hyperactive transposase gene, on a mini-F plasmid for stable maintenance in Escherichia coli K-12. Sixteen regions of chromosome V showing ARS activity were identified, of which 12 were newly found in this work. Thus, the procedure will be useful for systematic genomic scale analysis of ARSs in yeast and related organisms in which ordered clone banks have been established. The average distance between adjacent ARS-containing regions was approximately 40 kb. Two-dimensional gel electrophoretic analysis of chromosome replication indicated that one of the newly identified ARSs was functional as an actual in situ replication origin, at least under the conditions employed.