Noriyuki Tsujimura

Enhancement of magnetic particle production by nitrate and succinate fed-batch culture of Magnetospirillum sp. AMB-1

Magnetospirillum sp. AMB-1 is a magnetic bacterium, which is capable of growing under air atmosphere. This bacterium was employed to make bacterial magnetic particles (BMPs). AMB-1 only makes BMPs during logarithmic growth phase under anaerobic conditions. Since it requires nitrate as a nitrogen source, control of nitrate concentration in the medium was necessary. The fed-batch culture of AMB-1 was carried out by adding nitric acid and succinate as nitrogen and carbon source respectively. One liter of AMB-1 culture produced 0.34 g of dry cells and 4.5 mg of BMPs. BMP production by AMB-1 cultivated in the fed-batch culture was found to be seven times higher than that cultivated in the batch culture.

Respiratory inhibitors of a magnetic bacterium Magnetospirillum sp. AMB-1 capable of growing aerobically

Respiratory inhibitors of a magnetic bacterium Magnetospirillum sp. AMB-1, which is able to grow aerobically, were investigated using a microbial electrode system. The respiration of strain AMB-1 was inhibited by 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), KCN and dicumarol. Strain AMB-1 cannot grow in the presence of these inhibitors under aerobic conditions. On the other hand, strain AMB-1 can grow and form magnetite (Fe3O4) particles with HQNO and KCN under anaerobic conditions. Growth and magnetite formation of strain AMB-1 were reduced by dicumarol, which also inhibited iron reduction under anaerobic conditions, whereas iron reduction was not inhibited by HQNO and KCN.

Leukocyte counting from a small amount of whole blood using a size-controlled microcavity array.

Absolute counting of total leukocytes or specific subsets within small amounts of whole blood is difficult due to a lack of techniques that enable separation of all leukocytes from limited amounts of whole blood. In this study, a microfluidic device equipped with a size‐controlled microcavity array for highly efficient separation of leukocytes from submicroliters of whole blood was developed. The microcavity array can separate leukocytes from whole blood based on differences in the size and deformability between leukocytes and other blood cells. Leukocytes recovered on aligned microcavities were continuously processed for image‐based immunophenotypic analysis. Our device successfully recovered over 90% of leukocytes in 1 µL of whole blood without pretreatment such as density gradient centrifugation or erythrocyte lysis. In addition, the proposed system successfully performed absolute enumeration of human CD4+ and CD8+ leukocytes from 1 µL of whole blood, and the obtained data showed good correlation with conventional flow cytometric analysis. Our microfluidic device has great potential as a tool for a point‐of‐care leukocyte analysis system. Biotechnol. Bioeng. 2012; 109:2017–2024.

A novel method for isolation of magnetic bacteria without magnetic collection using magnetotaxis

Abstract A novel method was developed for isolating magnetic bacteria without magnetic collection using magnetotaxis. The method consists of incubation of sediments, enrichment of bacteria in the medium, isolation of enriched bacteria by colony formation, and optimization of conditions for growth and synthesis of magnetic particles. The water column above natural sediment, incubated at 25°C under dim light, and containing many species of bacteria, was employed as the inoculum. Collection of magnetic bacteria using magnets was not carried out. Ferric quinate was used as the main iron source in the liquid isolation medium. Due to iron sulfide precipitation, formation of black crystals was observed in the enriched culture of magnetic bacteria. Magnetic bacteria were purified by colony formation from enriched cultures which formed the black crystals. Culturing condition was optimized by addition of appropriate nutrients which behave as electron acceptors or donors. This method allows isolation of non-motile and non- or weakly-magnetotactic bacteria, which would not accumulate in the presence of an applied magnetic field. A sulphate-reducing magnetic anaerobe, RS-1, which is weakly magnetotactic, was isolated by this method. In addition, the successful isolation of RS-1 by this method suggests the presence of magnetic bacteria which exist in a non-magnetic state in sediments.

Magnetic control of bacterial magnetite-myosin conjugate movement on actin cables

Abstract The control of actin-myosin movement using a bacterial magnetic particle (BMP) which was synthesized intracellularly by magnetic bacteria was investigated. Myosin immobilized on the BMP ( myosin-BMP conjugate) was prepared with heterobifunctional reagents, Sulfo-LC-SPDP (sulfosuccinimidyl 6-(3′-(2-pyridyldithio)-propionamido)hexanoate) and Sulfo-SMCC (sulfosuccinimidyl 4-( N -maleimidomethyl)cyclohexane-1-carboxylate). When a magnetic force was applied in the same direction as the myosin-BMP conjugate movement (negative magnetic force) at 22–23°C, the velocity increased from 2.0 μm s −1 to 7.5 μm s −1 . When a magnetic force was applied in the opposite direction to the myosin-BMP conjugate movement ( positive magnetic force ), the velocity decreased from 10.7 μ s −1 to 0 μm s −1 . These results indicate that the sliding force of the myosin-BMP conjugate caused by the actin-myosin interaction can be controlled using a neodymium-boron (Nd-B) magnet. It will be possible to develop a nano-sized actuator that can be controlled by the external magnetic force. This system could also be applicable to analysis of the dynamics of a single myosin molecule.

Assessment of Benzene-Induced Hematotoxicity Using a Human-Like Hematopoietic Lineage in NOD/Shi-scid/IL-2Rγnull Mice

Despite recent advancements, it is still difficult to evaluate in vivo responses to toxicants in humans. Development of a system that can mimic the in vivo responses of human cells will enable more accurate health risk assessments. A surrogate human hematopoietic lineage can be established in NOD/Shi-scid/IL-2Rγnull (NOG) mice by transplanting human hematopoietic stem/progenitor cells (Hu-NOG mice). Here, we first evaluated the toxic response of human-like hematopoietic lineage in NOG mice to a representative toxic agent, benzene. Flow cytometric analysis showed that benzene caused a significant decrease in the number of human hematopoietic stem/progenitor cells in the bone marrow and the number of human leukocytes in the peripheral blood and hematopoietic organs. Next, we established chimeric mice by transplanting C57BL/6 mouse-derived bone marrow cells into NOG mice (Mo-NOG mice). A comparison of the degree of benzene-induced hematotoxicity in donor-derived hematopoietic lineage cells within Mo-NOG mice indicated that the toxic response of Hu-NOG mice reflected interspecies differences in susceptibilities to benzene. Responses to the toxic effects of benzene were greater in lymphoid cells than in myeloid cells in Mo-NOG and Hu-NOG mice. These findings suggested that Hu-NOG mice may be a powerful in vivo tool for assessing hematotoxicity in humans, while accounting for interspecies differences.

Comprehensive evaluation of leukocyte lineage derived from human hematopoietic cells in humanized mice

Recently, humanized animals whereby a part of the animal is biologically engineered using human genes or cells have been utilized to overcome interspecific differences. Herein, we analyzed the detail of the differentiation states of various human leukocyte subpopulations in humanized mouse and evaluated comprehensively the similarity of the leukocyte lineage between humanized mice and humans. Humanized mice were established by transplanting human CD34(+) cord blood cells into irradiated severely immunodeficient NOD/Shi-scid/IL2Rγ(null) (NOG) mice, and the phenotypes of human cells contained in bone marrow, thymus, spleen and peripheral blood from the mice were analyzed at monthly intervals until 4 months after cell transplantation. The analysis revealed that transplanted human hematopoietic stem cells via the caudal vein homed and engrafted themselves successfully at the mouse bone marrow. Subsequently, the differentiated leukocytes migrated to the various tissues. Almost all of the leukocytes within the thymus were human cells. Furthermore, analysis of the differentiation states of human leukocytes in various tissues and organs indicated that it is highly likely that the human-like leukocyte lineage can be developed in mice.

Monitoring of benzene-induced hematotoxicity in mice by serial leukocyte counting using a microcavity array

Monitoring of hematotoxicity, which requires serial blood collection, is difficult to carry out in small animals due to a lack of non-invasive, individual animal-appropriate techniques that enable enumeration of leukocyte subsets from limited amounts of whole blood. In this study, a microfluidic device equipped with a microcavity array that enables highly efficient separation of leukocytes from submicroliters of whole blood was applied for hematotoxicity monitoring in mice. The microcavity array can specifically separate leukocytes from whole blood based on differences in the size and deformability between leukocytes and other blood cells. Mouse leukocytes recovered on aligned microcavities were continuously processed for image-based immunophenotypic analysis. Our device successfully recovered almost 100% of mouse leukocytes in 0.1 μL of whole blood without the effect of serial blood collection such as changes in body weight and total leukocyte count. We assessed benzene-associated hematotoxicity in mice using this system. Mice were administered with benzene once daily and the depression of leukocyte numbers induced in individual mice was successfully monitored from tail vein blood collected every other day for 2 weeks. Serial monitoring of the leukocyte number in individual mice will contribute to the understanding of hematotoxicity and reduction of the number of animal experiment trials.

Production of a Protein (Enzyme, Antibody, Protein A)-Magnetite Complex by Genetically Engineered Magnetic Bacteria Magnetospirillum Sp. AMB-1

Magnetospirillum sp. AMB-1 is a magnetic bacterium which synthesizes intracellular particles of magnetite (Fe3O4). A genomic DNA fragment required for the synthesis of magnetic particles was previously isolated from this strain. The complete nucleotide sequence of this fragment has been determined by us. An open reading frame (ORF), designated magA, encodes a polypeptide which represents an iron transport protein. Intracellular localization of the MagA protein was studied using magA—luc fusion proteins. The luc gene was cloned downstream of the magA hydrophilic C-terminal domain. The fusion protein was detected on the surface of the lipid bilayer covering the magnetic particles. The MagA protein on the bacterial magnetic particle (BMP) was detected by immunoassay using an anti-luciferase antibody. The N- and C-termini of MagA protein were found outside the BMP membrane. These results show the utility of magA fusions for detecting multi functional proteins on BMP. Recombinant protein-BMP complex production has been carried out using the fed-batch culture by adding nitric acid and succinate as nitrogen and carbon source.