Magnetic separation of CD14+ cells using antibody binding with protein A expressed on bacterial magnetic particles for generating dendritic cells

Herein the potential of a highly efficient cell separation system using bacterial magnetic particles expressing protein A (protein A-BacMPs) was demonstrated. Protein A was expressed on BacMPs using the transmembrane proteins Mms13 and MagA as anchor molecules. The evaluations of the numbers of bound antibody molecules and binding capabilities of the protein A-BacMPs using Mms13 indicated that the antibodies were efficiently introduced into protein A-BacMPs using Mms13 in comparison to MagA. In addition, the recovery ratio of the target cells on the magnetic cell separation system was enhanced by using protein A-BacMPs with Mms13. Using positive selection against peripheral blood mononuclear cells, the CD14(+) cells were separated at a purity of more than 99% by protein A-BacMPs using Mms13. Furthermore, in the evaluation of the influence of protein A-BacMPs on the separated cells, the CD14(+) cells separated using protein A-BacMPs and were successfully differentiated into dendritic cells.     

Magnetic cell separation using nano-sized bacterial magnetic particles with reconstructed magnetosome membrane

Magnetic nanoparticles produced by magnetotactic bacterium, bacterial magnetic particles (BacMPs), covered with a lipid bilayer membrane (magnetosome membrane) can be used to separate specific target cells from heterogeneous mixtures because they are easily manipulated by magnets and it is easy to display functional proteins on their surface via genetic engineering. Despite possessing unique and valuable characteristics, the potential toxicity of BacMPs to the separated cells has not been characterized in detail. Here, a novel technique was developed for the reconstruction of magnetosome membrane of BacMPs expressing protein A (protein A-BacMPs) to reduce cytotoxicity and the newly developed nanomaterial was then used for magnetic cell separation. The development of the magnetosome membrane-reconstructed protein A-BacMP was based on the characteristics of the Mms13 anchor protein, which strongly binds to the magnetite surface of BacMPs. Treatment of protein A-BacMPs with detergents removed contaminating proteins but did not affect retention of Mms13-protein A fusion proteins. The particle surfaces were then reconstructed with phosphatidylcholine. The protein A-BacMPs containing reconstructed magnetosome membranes remained dispersible and retained the ability to immobilize antibody. In addition, they contained few membrane surface proteins and endotoxins, which were observed on non-treated protein A-BacMPs. Magnetic separation of monocytes and B-lymphocytes from the peripheral blood was achieved with high purity using magnetosome membrane-reconstructed protein A-BacMPs.     

Nano‐sized bacterial magnetic particles displaying pyruvate phosphate dikinase for pyrosequencing

There is a high demand for inexpensive and high‐throughput DNA sequencing technologies in molecular biology and applied biosciences. In this study, novel nano‐sized magnetic particles displaying enzymes for pyrosequencing, a rather novel bioluminometric DNA sequencing method based on the sequencing‐by‐synthesis principle by employing a cascade of several enzymatic reactions, was developed. A highly thermostable enzyme, pyruvate phosphate dikinase (PPDK) which converts PPi to ATP was successfully expressed onto bacterial magnetic particles (BacMPs) using a novel protein display system of Magnetospirillum magneticum AMB‐1. The enzymatic stability of BacMPs displaying PPDK (PPDK‐BacMPs) to pH and temperature was evaluated and its broad range of properties was shown. Subsequently, PPDK‐BacMPs were applied in pyrosequencing and a target oligonucleotide was successfully sequenced. The PPDK enzyme displayed on BacMPs was shown to be recyclable in each sequence reaction as they can be manipulated by magnetic force. It was concluded that nano‐sized PPDK‐BacMPs are useful for the scale down of pyrosequencing reaction volumes, thus, permitting high‐throughput. The recycling of enzymes was also shown to be promising and applicable for the development of an inexpensive DNA sequencing at a low running cost. Biotechnol. Bioeng. 2009;103: 130–137. © 2008 Wiley Periodicals, Inc.     

Quantification of non‐specific binding of magnetic micro‐ and nanoparticles using cell tracking velocimetry: Implication for magnetic cell separation and detection

The maturation of magnetic cell separation technology places increasing demands on magnetic cell separation performance. While a number of factors can cause sub‐optimal performance, one of the major challenges can be non‐specific binding of magnetic nano‐ or microparticles to non‐targeted cells. Depending on the type of separation, this non‐specific binding can have a negative effect on the final purity, the recovery of the targeted cells, or both. In this work, we quantitatively demonstrate that non‐specific binding of magnetic nanoparticles can impart a magnetization to cells such that these cells can be retained in a separation column and thus negatively impact the purity of the final product and the recovery of the desired cells. Through experimental data and theoretical arguments, we demonstrate that the number of MACS magnetic particles needed to impart a magnetization that is sufficient to cause non‐targeted cells to be retained in the column to be on the order of 500–1,000 nanoparticles. This number of non‐specifically bound particles was demonstrated experimentally with an instrument, cell tracking velocimeter, CTV, and it is demonstrated that the sensitivity of the CTV instrument for Fe atoms contained in magnetic nanoparticles on the order of 1 × 10^?15 g/mL of Fe. Biotechnol. Bioeng. 2010;105: 1078–1093. © 2009 Wiley Periodicals, Inc.     

Bioengineering of bacterial magnetic particles and their applications in biotechnology

Magnetic particles have attracted much attention for their versatile uses in biotechnology, especially in medical applications. The major advantage of magnetic particles is that they can be easily manipulated by magnetic forces. Magnetotactic bacteria synthesize nano-sized biomagnetites, otherwise known as bacterial magnetic particles (BacMPs) that are individually enveloped by a lipid bilayer membrane. The mechanisms of BacMP synthesis have been analyzed by genomic, proteomic, and bioinformatic approaches. Based on those studies in Magnetospirillum magneticum AMB-1, functional nanomaterials have been designed and produced. Through genetic engineering, functional proteins such as enzymes, antibodies, and receptors have been successfully displayed on BacMPs. These functional BacMPs have been utilized in various biosensors and bio-separation processes. Here, recent papers and patents for bioengineering of BacMPs and their applications in biotechnology are reviewed. The elucidation of the mechanism of magnetic particle synthesis has provided a roadmap for the design of novel biomaterials that can play useful roles in multiple disciplinary fields.     

Functional expression of an scFv on bacterial magnetic particles by in vitro docking

A Gram-negative, magnetotactic bacterium, Magnetospirillum magneticum AMB-1 produces nano-sized magnetic particles (BacMPs) in the cytoplasm. Although various applications of genetically engineered BacMPs have been demonstrated, such as immunoassay, ligand–receptor interaction or cell separation, by expressing a target protein on BacMPs, it has been difficult to express disulfide-bonded proteins on BacMPs due to lack of disulfide-bond formation in the cytoplasm. Here, we propose a novel dual expression system, called in vitro docking, of a disulfide-bonded protein on BacMPs by directing an immunoglobulin Fc-fused target protein to the periplasm and its docking protein ZZ on BacMPs. By in vitro docking, an scFv–Fc fusion protein was functionally expressed on BacMPs in the dimeric or trimeric form. Our novel disulfide-bonded protein expression system on BacMPs will be useful for efficient screening of potential ligands or drugs, analyzing ligand–receptor interactions or as a magnetic carrier for affinity purification.     

Novel Method for Selection of Antimicrobial Peptides from a Phage Display Library by Use of Bacterial Magnetic Particles

Antimicrobial peptides were isolated from a phage display peptide library using bacterial magnetic particles (BacMPs) as a solid support. The BacMPs obtained from “Magnetospirillum magneticum” strain AMB-1 consist of pure magnetite (50 to 100 nm in size) and are covered with a lipid bilayer membrane derived from the invagination of the inner membrane. BacMPs are easily purified from a culture of magnetotactic bacteria by magnetic separation. Approximately 4 × 10¹⁰ PFU of the library phage (complexity, 2.7 × 10⁹) was reacted with BacMPs. The elution of bound phages from BacMPs was performed by disrupting its membrane with phospholipase D treatment. Six candidate peptides, which were highly cationic and could bind onto the BacMP membrane, were obtained. They exhibited antimicrobial activity against Bacillus subtilis but not against Escherichia coli and Saccharomyces cerevisiae. The amino acid substitution of the selected peptide, KPQQHNRPLRHK (peptide 6-7), to enhance the hydrophobicity resulted in obvious antimicrobial activity against all test microorganisms. The present study shows for the first time that a magnetic selection of antimicrobial peptides from the phage display peptide library was successfully achieved by targeting the actual bacterial inner membrane. This BacMP-based method could be a promising approach for a high-throughput screening of antimicrobial peptides targeting a wide range of species.     

Inducible Expression of Transmembrane Proteins on Bacterial Magnetic Particles in Magnetospirillum magneticum AMB-1

Bacterial magnetic particles (BacMPs) produced by the magnetotactic bacterium Magnetospirillum magneticum AMB-1 are used for a variety of biomedical applications. In particular, the lipid bilayer surrounding BacMPs has been reported to be amenable to the insertion of recombinant transmembrane proteins; however, the display of transmembrane proteins in BacMP membranes remains a technical challenge due to the cytotoxic effects of the proteins when they are overexpressed in bacterial cells. In this study, a tetracycline-inducible expression system was developed to display transmembrane proteins on BacMPs. The expression and localization of the target proteins were confirmed using luciferase and green fluorescent protein as reporter proteins. Gene expression was suppressed in the absence of anhydrotetracycline, and the level of protein expression could be controlled by modulating the concentration of the inducer molecule. This system was implemented to obtain the expression of the tetraspanin CD81. The truncated form of CD81 including the ligand binding site was successfully displayed at the surface of BacMPs by using Mms13 as an anchor protein and was shown to bind the hepatitis C virus envelope protein E2. These results suggest that the tetracycline-inducible expression system described here will be a useful tool for the expression and display of transmembrane proteins in the membranes of BacMPs.Transmembrane proteins play critical roles in cellular metabolism, participating in processes such as ion transport, nutrient uptake, signal transduction, and intercellular communication. As evidence of the essential functions of these proteins, more than half of all drug targets have been shown to be transmembrane proteins, and the analysis of the interactions of transmembrane proteins and their ligands is one of the most promising avenues for the discovery of new drug candidates. As a means of producing sufficient amounts of transmembrane proteins for binding analyses, heterologous protein expression systems have been developed using Escherichia coli (10), yeast (16), insect, and mammalian (4) cells as hosts. Transmembrane proteins generally are expressed at low levels and are extremely hydrophobic, rendering the analysis of interactions with ligands very difficult. In all cases, the analysis of membrane proteins requires a lipid or similar synthetic environment to maintain the native structure and function of the proteins. The purification of transmembrane proteins from cells frequently is time-consuming and typically results in the loss of the proteins’ native conformation.Magnetospirillum magneticum AMB-1 synthesizes intracellular nanosized bacterial magnetic particles (BacMPs; 50 to 100 nm); these are surrounded by a lipid bilayer membrane and exhibit strong ferrimagnetism. Functional soluble proteins have been expressed on BacMP surfaces through gene fusion techniques (11, 21, 24, 27) using BacMP membrane proteins (MagA, Mms16, and Mms13) as anchor proteins; this approach permits heterologous proteins to be localized efficiently and oriented appropriately on BacMPs. In a previous report, we demonstrated the successful display of the D1 dopamine receptor, a G protein-coupled receptor possessing seven transmembrane domains, on BacMPs. Mms16-D1, an dopamine receptor fusion protein, was expressed under the mms16 promoter, and a ligand-binding assay was performed (28). The assembly of transmembrane proteins on magnetic particles provides significant advantages for binding assays, including the easing of the purification of target proteins from bacterial cells without the loss of native conformation and the availability of a fully automated bioassay using robotic magnetic separation. Despite these advantages, there are not enough studies for the overexpression of transmembrane proteins other than the D1 dopamine receptor in M. magneticum AMB-1 because of its difficulty. In other host cells, a system for controlling gene expression has been employed to overcome its difficulty, and some successful efforts had achieved this for crystal structure analysis (5, 15, 18). The lack of these systems for M. magneticum has hampered the extension of this application to other transmembrane proteins.In this study, the tetracycline-inducible expression system was adapted for displaying transmembrane proteins on BacMPs in M. magneticum AMB-1. Expression vectors carrying the tetracycline repressor gene (tetR) and the target gene under the control of a strong promoter and the tetracycline operator (tetO) sequence were constructed, and the function of the system was evaluated using reporter genes. Finally, this system was applied to the overexpression of the transmembrane protein, tetraspanin CD81. This is the first report of an inducible expression system in M. magneticum, and it the demonstrates efficient display of a transmembrane protein at the surface of BacMPs.     

Binding affinities/avidities of antibody-antigen interactions: quantification and scale-up implications

Bioaffinity interactions have been, and continue to be, successfully adapted from nature for use in separation and detection applications. It has been previously reported that the magnetophoretic mobility of labeled cells show a saturation type phenomenon as a function of the concentration of the free antibody-magnetic nanoparticle conjugate which is consistent with other reports of antibody-fluorophore binding. Starting with the standard antibody-antigen relationship, a model was developed which takes into consideration multi-valence interactions, and various attributes of flow cytometry (FCM) and cell tracking velocimetry (CTV) measurements to determine both the apparent dissociation constant and the antibody-binding capacity (ABC) of a cell. This model was then evaluated on peripheral blood lymphocytes (PBLs) labeled with anti CD3 antibodies conjugated to FITC, PE, or DM (magnetic nanoparticles). Reasonable agreements between the model and the experiments were obtained. In addition, estimates of the limitation of the number of magnetic nanoparticles that can bind to a cell as a result of steric hinderance was consistent with measured values of magnetophoretic mobility. Finally, a scale-up model was proposed and tested which predicts the amount of antibody conjugates needed to achieve a given level of saturation as the total number of cells reaches 10(10), the number of cells needed for certain clinical applications, such as T-cell depletions for mismatched bone marrow transplants.     

Metallothionein in human immunomagnetically selected CD34+ haematopoietic progenitor cells

Human umbilical CD34⁺ immature haematopoietic cells were rapidly and efficiently obtained from light density MNC (mononuclear cells) by MACS (magnetic cell sorting). An ex vivo expanded population of CD34⁺ was cultured in serum‐free medium supplemented with cytokines FL (flt3 ligand), SCF (stem cell factor) and TPO (thrombopoietin) in order to obtain a sufficient number of CD34⁺ cells. CD34⁺ cells expanded from cord blood for 7 days were demonstrated to increase in the absolute number of CD34⁺ cells by 5.12±2.47‐fold (mean±S.D., n=3). Flow cytometric analysis demonstrated that the percentage of CD34 antigen expression after expansion of the culture was 97.81±1.07%, whereas it was 69.39±10.37% in none‐expanded CD34⁺ cells (mean±S.D., n=3), thus defining a system that allowed extensive amplification accompanied by no maturation. MTs (metallothioneins), low molecular weight, cysteine‐rich metal‐binding proteins, exhibit various functions, including metal detoxification and homoeostasis. We here examined the expression pattern of functional members of the MT gene family in immature CD34⁺ cells and compared it with more mature CD34^? cells in order to strengthen the proposed function of MT in differentiation. Cells were cultured in RPMI 1640 medium, with or without different zinc supplements for 24 h. Relative quantitative expression of MT isogenes in the mature CD34^? cells was higher than in the immature CD34⁺ cells. IHC (immunohistochemical staining) revealed an increased MT protein biosynthesis in CD34^? cells, greater than in CD34⁺ cells. Therefore, the role of MT in differentiation of human haematopoietic progenitor cells from human cord blood is reported for the first time.     

Involvement of HAb18G/CD147 in T cell activation and immunological synapse formation

HAb18G/CD147, a glycoprotein of the immunoglobulin super‐family (IgSF), is a T cell activation‐associated molecule. In this report, we demonstrated that HAb18G/CD147 expression on both activated CD4⁺ and CD8⁺ T cells was up‐regulated. In vitro cross‐linking of T cells with an anti‐HAb18G/CD147 monoclonal antibody (mAb) 5A12 inhibited T cells proliferation upon T cell receptor stimulation. Such co‐stimulation inhibited T cell proliferation by down‐regulating the expression of CD25 and interleukin‐2 (IL‐2), decreased production of IL‐4 but not interferon‐γ. Laser confocal imaging analysis indicated that HAb18G/CD147 was recruited to the immunological synapse (IS) during T cell activation; triggering HAb18G/CD147 on activated T cells by anti‐HAb18G/CD147 mAb 5A12 strongly dispersed the formation of the IS. Further functional studies showed that the ligation of HAb18G/CD147 with mAb 5A12 decreased the tyrosine phosphorylation and intracellular calcium mobilization levels of T cells. Through docking antibody–antigen interactions, we demonstrated that the function of mAb 5A12 is tightly dependent on its specificity of binding to N‐terminal domain I, which plays pivotal role in the oligomerization of HAb18G/CD147. Taken together, we provide evidence that HAb18G/CD147 could act as a co‐stimulatory receptor to negatively regulate T cell activation and is functionally linked to the formation of the IS.     

One‐step magnetic modification of yeast cells by microwave‐synthesized iron oxide microparticles

Baker's yeast (Saccharomyces cerevisiae) cells were magnetically modified with magnetic iron oxide particles prepared by microwave irradiation of iron(II) sulfate at high pH. The modification procedure was very simple and fast. Both non‐cross‐linked and glutaraldehyde cross‐linked magnetic cells enabled efficient sucrose conversion into glucose and fructose, due to the presence of active intracellular invertase. The prepared magnetic whole‐cell biocatalyst was stable; almost the same catalytic activity was observed after 1‐month storage at 4°C. Simple magnetic separation and stability of the developed biocatalyst enabled its reusability without significant loss of enzyme activity.

Significance and Impact of the Study

Magnetic whole yeast cell biocatalyst containing intracellular invertase in its natural environment has been prepared. Magnetic properties enable its easy separation from reaction mixture. Magnetically modified Saccharomyces cerevisiae cells have been used for invert sugar production, hydrolysing sucrose into glucose and fructose. The described magnetization procedure employing microwave‐synthesized iron oxide microparticles is a low‐cost and easy‐to‐perform alternative to already existing magnetization techniques.     

Magnetic Precipitation: A New Platform for Protein Purification

One of the trends in downstream processing comprises the use of “anything‐but‐chromatography” methods to overcome the current downfalls of standard packed‐bed chromatography. Precipitation and magnetic separation are two techniques already proven to accomplish protein purification from complex media, yet never used in synergy. With the aim to capture antibodies directly from crude extracts, a new approach combining precipitation and magnetic separation is developed and named as affinity magnetic precipitation. A precipitation screening, based on the Hofmeister series, and a commercial precipitation kit are tested with affinity magnetic particles to assess the best condition for antibody capture from human serum plasma and clarified cell supernatant. The best conditions are obtained when using PEG3350 as precipitant at 4 °C for 1 h, reaching 80% purity and 50% recovery of polyclonal antibodies from plasma, and 99% purity with 97% recovery yield of anti‐TNFα mAb from cell supernatants. These results show that the synergetic use of precipitation and magnetic separation can represent an alternative for the efficient capture of antibodies.     

In Vivo Biotinylation of Bacterial Magnetic Particles by a Truncated Form of Escherichia coli Biotin Ligase and Biotin Acceptor Peptide

Escherichia coli biotin ligase can attach biotin molecules to a lysine residue of biotin acceptor peptide (BAP), and biotinylation of particular BAP-fused proteins in cells was carried out by coexpression of E. coli biotin ligase (in vivo biotinylation). This in vivo biotinylation technology has been applied for protein purification, analysis of protein localization, and protein-protein interaction in eukaryotic cells, while such studies have not been reported in bacterial cells. In this study, in vivo biotinylation of bacterial magnetic particles (BacMPs) synthesized by Magnetospirillum magneticum AMB-1 was attempted by heterologous expression of E. coli biotin ligase. To biotinylate BacMPs in vivo, BAP was fused to a BacMP surface protein, Mms13, and E. coli biotin ligase was simultaneously expressed in the truncated form lacking the DNA-binding domain. This truncation-based approach permitted the growth of AMB-1 transformants when biotin ligase was heterologously expressed. In vivo biotinylation of BAP on BacMPs was confirmed using an alkaline phosphatase-conjugated antibiotin antibody. The biotinylated BAP-displaying BacMPs were then exposed to streptavidin by simple mixing. The streptavidin-binding capacity of BacMPs biotinylated in vivo was 35-fold greater than that of BacMPs biotinylated in vitro, where BAP-displaying BacMPs purified from bacterial cells were biotinylated by being mixed with E. coli biotin ligase. This study describes not only a simple method to produce biotinylated nanomagnetic particles but also a possible expansion of in vivo biotinylation technology for bacterial investigation.Biotin/streptavidin binding is the strongest noncovalent interaction known in nature (K_d [dissociation constant], ∼10⁻¹⁵ M) (10), and this tight binding is one of the most general tools for biological research and has been widely used for biomolecular detection (11, 12), immobilization (14, 19), and recovery (15). Therefore, it is of great significance to biotinylate biomolecules, in particular, proteins without functional inhibition. For this purpose, the method for site-selective biotinylation of proteins had been developed using biotin ligase. Biotin ligase catalyzes the posttranslational biotinylation of biotin enzymes, such as acetyl coenzyme A (acetyl-CoA) carboxylase, and introduces biotin into a specific lysine residue of a biotin carboxyl carrier protein (BCCP), a subunit of biotin enzymes (13). In early studies, BCCP (∼100 amino acid residues) had been fused with the proteins of interest for biotinylation by biotin ligase (7); however, there was a concern that fused BCCP might disrupt the function of target proteins. Recently, biotin acceptor peptides (BAPs) had replaced BCCP due to the advantage of small size. BAPs, with 15 to 23 amino acid residues, were screened from a peptide library as peptide tags biotinylated by Escherichia coli biotin ligase (4, 25). BAP-fused proteins can be biotinylated outside the cells by adding biotin and purified E. coli biotin ligase with Mg²⁺ and ATP (in vitro biotinylation). Furthermore, it is also possible to biotinylate BAP-fused proteins inside the cells with coexpression of E. coli biotin ligase (in vivo biotinylation) because BAP is specifically recognized only by E. coli biotin ligase. This in vivo biotinylation technology has been applied in eukaryotic cells to purify the proteins by using streptavidin-immobilized resin (8, 24, 28), because biotin/streptavidin interaction permits stringent washing to eliminate the nonspecific binding. Specific biotinylation can be applied also for protein localization analysis. Using fluorophore- or gold nanoparticle-labeled streptavidin, biotinylated proteins were clearly observed in a previous study (27). Recently, a novel technique to detect protein-protein interaction by fusing BAP and biotin ligase was developed by Ting''s group. BAP and biotin ligase were fused to different two proteins, and then the interaction of these proteins was successfully evaluated via biotinylation of BAP (9). In vivo biotinylation technology using heterologously expressed E. coli biotin ligase should be equally useful for prokaryotes; however, such studies have not been reported for bacterial cells.Magnetospirillum magneticum AMB-1, a magnetotactic bacterium, synthesizes intracellular nanosized bacterial magnetic particles (BacMPs) of 50 to 100 nm; these are surrounded by a lipid bilayer membrane, possess a single magnetic domain of magnetite, and exhibit strong ferrimagnetism (18). Furthermore, functional proteins have been displayed on BacMP surfaces through gene fusion techniques (21, 30, 31). BacMP membrane proteins, including Mms13, were used as anchor proteins; this approach permits functional proteins to be localized efficiently and oriented appropriately on BacMPs (31). We recently reported a novel method for the simple production of biotin-labeled magnetic particles through protein display techniques, where introduction of the biotin moiety onto BacMPs was carried out by the endogenous biotin ligase (17). For the biotinylation of BacMPs, we screened the gene encoding BCCP in the AMB-1 genome and displayed it on the surface of BacMPs using an anchor protein, Mms13. BCCP-displaying BacMPs were biotinylated by endogenous AMB-1 biotin ligase in the cells with high efficiency. This in vivo modification approach could be applied for construction of BacMP-quantum dot nanocomposites toward multicolor labeling of cancer cells, where BCCP and antibody carrier protein (protein G) were simultaneously displayed in tandem (16). However, the size of BCCP, with 149 amino acid residues and a mass of 15.6 kDa, makes it rather large for use as a labeling tag. Although it would be preferable to use a smaller peptide, BAP, for the tag to minimize effects on the flanking proteins for future applications, BAP was not recognized and biotinylated by endogenous AMB-1 biotin ligase (17).In this study, in vivo biotinylation of BacMPs was attempted by heterologous expression of E. coli biotin ligase and Mms13-BAP fusion protein in AMB-1 cells. First, the method for effective expression of E. coli biotin ligase in bacterial cells was optimized. Then site-selective biotinylation of BAP on BacMPs was confirmed. Finally, the obvious advantage of in vivo biotinylation of BAP-displaying BacMPs compared with the in vitro biotinylation method was demonstrated.     

Large gradient high magnetic field affects the association of MACF1 with actin and microtubule cytoskeleton

The intense inhomogeneous magnetic fields acting on the diamagnetic materials naturally present in cells can generate strong magnetic forces. We have developed a superconducting magnet platform with large gradient high magnetic field (LG‐HMF), which can produce three magnetic force fields of ?1360, 0, and 1312 T²/m, and three corresponding apparent gravity levels, namely 0, 1, and 2‐g for diamagnetic materials. In this study, the effects of different magnetic force fields on osteoblast‐like cells (MG‐63 and MC3T3‐E1) viability, microtubule actin crosslinking factor 1 (MACF1) expression and its association with cytoskeleton were investigated. Results showed that cell viability increased to different degrees after exposure to 0 or 1‐g conditions for 24 h, but it decreased by about 30% under 2‐g conditions compared with control conditions. An increase in MACF1 expression at the RNA or protein level was observed in osteoblast‐like cells under the magnetic force field of ?1360 T²/m (0‐g) relative to 1312 T²/m (2‐g). Under control conditions, anti‐MACF1 staining was scattered in the cytoplasm and partially colocalized with actin filaments (AFs) or microtubules (MTs) in the majority of osteoblast‐like cells. Under 0‐g conditions, MACF1 labeling was concentrated at perinuclear region and colocalization was not apparent. The patterns of anti‐MACF1 labeling on MTs varied with MTs' changing under LG‐HMF environment. In conclusion, LG‐HMF affects osteoblast‐like cell viability, MACF1 distribution, expression, and its association with cytoskeleton to some extent. Bioelectromagnetics 30:545–555, 2009. © 2009 Wiley‐Liss, Inc.     

Exposure to a MRI‐type high‐strength static magnetic field stimulates megakaryocytic/erythroid hematopoiesis in CD34+ cells from human placental and umbilical cord blood

The biological response after exposure to a high‐strength static magnetic field (SMF) has recently been widely discussed from the perspective of possible health benefits as well as potential adverse effects. To clarify this issue, CD34⁺ cells from human placental and umbilical cord blood were exposed under conditions of high‐strength SMF in vitro. The high‐strength SMF exposure system was comprised of a magnetic field generator with a helium‐free superconducting magnet with built‐in CO₂ incubator. Freshly prepared CD34⁺ cells were exposed to a 5 tesla (T) SMF with the strongest magnetic field gradient (41.7 T/m) or a 10 T SMF without magnetic field gradient for 4 or 16 h. In the harvested cells after exposure to 10 T SMF for 16 h, a significant increase of hematopoietic progenitors in the total burst‐forming unit erythroid‐ and megakaryocytic progenitor cells‐derived colony formation was observed, thus producing 1.72‐ and 1.77‐fold higher than the control, respectively. Furthermore, early hematopoiesis‐related and cell cycle‐related genes were found to be significantly up‐regulated by exposure to SMF. These results suggest that the 10 T SMF exposure may change gene expressions and result in the specific enhancement of megakaryocytic/erythroid progenitor (MEP) differentiation from pluripotent hematopoietic stem cells and/or the proliferation of bipotent MEP. Bioelectromagnetics 30:280–285, 2009. © 2009 Wiley‐Liss, Inc.     

Killer cells induced by stimulation with allogeneic tumor cells and subsequent culture with recombinant interleukin-2

Summary Peripheral blood lymphocytes were cultured for 5 days with allogeneic tumor cells (allogeneic mixed lymphocyte/tumor cell culture), and subsequently cultured with recombinant interleukin-2 for 12 days. These cultured cells were found to be cytotoxic to autologous tumor cells. Results of two-color analysis using monoclonal antibodies to cell markers showed that more than 80% of their cultured cells were CD3⁺ cells, and CD4⁺ cells showed a higher distribution than CD8⁺ cells. However, CD8⁺ cells had a much higher killing activity with autologous tumor than did CD4⁺ cells, when estimated by an elimination study using monoclonal antibodies to T cell phenotypes and complement. The cold-target inhibition test showed that the cytotoxicity of these cells for autologous tumor cells was inhibited by unlabeled autologous tumor cells but not by unlabeled stimulator cells. Furthermore, about 40% of the cytotoxicity was suppressed by blocking of HLA class I antigen with a monoclonal antibody on autologous tumor cells. Thus, cytotoxic activity of lymphocytes to autologous tumor restricted by target cell HLA class I antigen is possibly induced by allogeneic tumor-stimulation.     

A novel technique for in situ aggregation of Gluconobacter oxydans using bio‐adhesive magnetic nanoparticles

Here, we present a novel technique to immobilize magnetic particles onto whole Gluconobacter oxydans in situ via a synthetic adhesive biomimetic material inspired by the protein glues of marine mussels. Our approach involves simple coating of a cell adherent polydopamine film onto magnetic nanoparticles, followed by conjugation of the polydopamine‐coated nanoparticles to G. oxydans which resulted in cell aggregation. After optimization, 21.3 mg (wet cell weight) G. oxydans per milligram of nanoparticle was aggregated and separated with a magnet. Importantly, the G. oxydan aggregates showed high specific activity and good reusability. The facile approach offers the potential advantages of low cost, easy cell separation, low diffusion resistance, and high efficiency. Furthermore, the approach is a convenient platform technique for magnetization of cells in situ by direct mixing of nanoparticles with a cell suspension. Biotechnol. Bioeng. 2012; 109: 2970–2977. © 2012 Wiley Periodicals, Inc.     

Screening HIV‐1 antigenic peptides as receptors for antibodies and CD4 in allosteric nanosensors

We have analyzed the suitability of six antigenic peptides from several HIV‐1 structural proteins (namely gp41, gp120, p17, and p24), as anti‐HIV‐1 antibody receptors in an allosteric enzymatic biosensor. These peptides were inserted in a solvent‐exposed surface of Escherichia coli (E. coli) beta‐galactosidase by means of conventional recombinant DNA technology. The resulting enzymes were tested to allosterically respond to sera from HIV‐1‐infected individuals. Only stretches from gp41 and gp120 envelope proteins were able to transduce the molecular contact signal in the presence of immunoreactive sera. Intriguingly, the enzyme displaying the CD4 binding site segment KQFINMWQEVGKAMYAPP was activated by soluble CD4, suggesting that it produces conformational modifications on the allosteric enzyme as those occurring during antibody‐promoted induced fit. This fact is discussed in the context of the design of smart protein drugs and markers targeted to CD4⁺ cells. Copyright © 2009 John Wiley & Sons, Ltd.     

Microvirin,a Novel α(1,2)-Mannose-specific Lectin Isolated from Microcystis aeruginosa,Has Anti-HIV-1 Activity Comparable with That of Cyanovirin-N but a Much Higher Safety Profile

Microvirin (MVN), a recently isolated lectin from the cyanobacterium Microcystis aeruginosa PCC7806, shares 33% identity with the potent anti-human immunodeficiency virus (HIV) protein cyanovirin-N (CV-N) isolated from Nostoc ellipsosporum, and both lectins bind to similar carbohydrate structures. MVN is able to inhibit infection by a wide variety of HIV-1 laboratory-adapted strains and clinical isolates of different tropisms and subtypes in peripheral blood mononuclear cells. MVN also inhibits syncytium formation between persistently HIV-1-infected T cells and uninfected CD4⁺ T cells and inhibits DC-SIGN-mediated HIV-1 binding and transmission to CD4⁺ T cells. Long term passaging of HIV-1 exposed to dose-escalating concentrations of MVN resulted in the selection of a mutant virus with four deleted high mannose-type glycans in the envelope gp120. The MVN-resistant virus was still highly sensitive to various other carbohydrate binding lectins (e.g. CV-N, HHA, GNA, and UDA) but not anymore to the carbohydrate-specific 2G12 monoclonal antibody. Importantly, MVN is more than 50-fold less cytotoxic than CV-N. Also in sharp contrast to CV-N, MVN did not increase the level of the activation markers CD25, CD69, and HLA-DR in CD4⁺ T lymphocytes, and subsequently, MVN did not enhance viral replication in pretreated peripheral blood mononuclear cells. Therefore, MVN may qualify as a useful lectin for potential microbicidal use based on its broad and potent antiviral activity and virtual lack of any stimulatory properties and cellular toxicity.