Production of recombinant human lysosomal acid lipase in Schizosaccharomyces pombe: development of a fed-batch fermentation and purification process

A fed-batch fermentation process has been developed to enable the production of large quantities of recombinant human lysosomal acid lipase (hLAL; EC 3.1.1.13), in Schizosaccharomyces pombe, for preclinical studies as a potential enzyme therapy drug. Recombinant S. pombe, clone ASP397-21, expressed enzymatically active hLAL in the secreted form. A feedback fed-batch system was used to determine the optimal feed rate of a 50% glucose solution used as the carbon source. The feed rate of the glucose solution was calculated by a computer-aided system according to the equation; F=q(sf)(VX)/S(in) (q(sf), specific substrate feed rate [gram substrate/gram dry cell weight/h]; V, volume of culture broth [l]; X, cell density [gram dry cell weight/l]; S(in), concentration of growth limiting substrate in feed solution [gram substrate/gram feed solution]). At the time of the initial consumption of glucose in the batch-phase culture, the nutrient supply was automatically initiated by means of monitoring the respiratory quotient change. The obtained profile of the feed rate was applied to the feed forward control fermentation. Finally, the cells were grown up to >50 g dry cell weight/l, and the hLAL expression level was approximately 16,000 U/l. Expressed hLAL protein was purified in a two-step process by hydrophobic interaction and anion exchange chromatographies. Purified recombinant hLAL exhibited a 90-150 kDa broad band upon SDS-PAGE with specific activity of about 300 U/mg. After endoglycosidase H treatment, the band converged to 45 kDa, equal to the calculated molecular weight, suggesting that hLAL produced in S. pombe was hyper-glycosylated. N-terminal analysis of de-glycosylated hLAL revealed that the signal sequence of hLAL was correctly processed in S. pombe.     

Formation of protein adducts of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine in cooked foods

Heterocyclic amines (HCAs) are mutagenic and carcinogenic compounds found in cooked meat and fish. Although HCAs are known to form adducts with protein after metabolic activation, adduct formation during cooking has not been elucidated. In this study, we showed that 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) is released from high molecular weight compounds by acid or enzymatic hydrolysis of cooked foods. Formation of free and protein adduct forms of PhIP was dependent on cooking temperature and time, and PhIP–protein adducts were estimated to form after formation of free PhIP. We also demonstrated that PhIP–protein adduct is formed by heating of PhIP and albumin as a model protein. A new adduct peak including [M+H]⁺ (m/z=225) of PhIP as a fragment ion was detected in the high molecular weight fraction of heat‐treated protein by LC–MS analysis. From model experiments by heating of PhIP and amino acids, the adduct was estimated to be produced by condensation of the amino group of PhIP and the carboxyl group of protein. PhIP–protein adducts were detected in several cooked meat and fish at ng/g food level as PhIP content. These results suggest that food‐borne protein adducts of HCAs may influence human HCA exposure and carcinogenic risk.     

3D reconstruction considering calculation time reduction for linear trajectory shooting and accuracy verification with simulator

Artificial Life and Robotics - Photogrammetry is a three-dimensional (3D) reconstruction from images. In photogrammetry, when each image captures the features of the target object for 3D...     

Effect of meso Positioned Substituents on the Stability and Photodynamic Activity of Lipid-Membrane-Incorporated Porphyrin Derivatives

In this study we prepared aqueous solutions of lipid-membrane incorporated tetraarylporphyrins and tetrapyridylporphyrin (LMIPors) by the injection method using dimethyl sulfoxide. The porphyrins with proton-donor groups at the meso position afforded stable aqueous solutions of LMIPors. However, although tetrakis(carboxyphenyl)porphyrin was scarcely incorporated in lipid membranes, it was soluble in water. Among these LMIPors, the photodynamic activity of tetrakis(hydroxyphenyl)porphyrin was higher than that of tetrakis(aminophenyl)porphyrin. This was attributed to the self-aggregation of a part of tetrakis(aminophenyl)porphyrin in the liposomes, which induced self-quenching and the consequent decrease of its photodynamic activity.     

Phospholipid-Coated Boronic Oxide Nanoparticles as Boron Agents for Boron Neutron Capture Therapy

Minimally invasive boron neutron capture therapy (BNCT) is an elegant approach for cancer treatment. The highly selective and efficient deliverability of boron agents to cancer cells is the key to maximizing the therapeutic benefits of BNCT. In addition, enhancement of the frequencies to achieve boron neutron capture reaction is also significant in improving therapeutic efficacy by providing a highly concentrated boron agent in each boron nanoparticle. As the density of the thermal neutron beam remains low, it is unable to induce high-efficiency cell destruction. Herein, we report phospholipid-coated boronic oxide nanoparticles as agents for BNCT that can provide a highly concentrated boron atom in each nanoparticle. The current system exhibited in vitro BNCT activity seven times higher than that of commercial boron agents. Furthermore, the system could penetrate cancer spheroids deeply, efficiently suppressing thermal neutron irradiation-induced growth.     

Increase of inductance ratio of flat type fault current limiter with high‐Tc superconducting disk

Fault current limiters (FCLs) are expected to reduce fault current and improve stability of power systems. A flat type fault current limiter consisting of the pancake primary winding and a high‐T_c superconducting (HTS) disk was proposed as a modified version of a conventional magnetic shield type of the FCL with the superconducting cylinder. The flat type FCL has the advantage of being manufactured smaller than the cylinder type FCL. It is pointed out, from a magnetic field analysis, that the volume of the flat type FCL is smaller than that of the cylinder type FCL with same magnitude of the limiting inductance. Further, the magnetic field analysis suggests that a high inductance ratio can be realized by radially enlarging both the primary winding and the HTS disk, stacking the FCL modules in layers and sandwiching the primary winding with superconducting disks. © 2006 Institute of Electrical Engineers of Japan. Published by John Wiley & Sons, Inc.