Kazuto Ikemoto

DNA Introduction into Living Cells by Water Droplet Impact with an Electrospray Process

Electrospraying is performed by applying a high voltage to a capillary tip. A highly charged droplet at the capillary tip splits into microor nanoscale droplets, and a charged fineliquid aerosol is accelerated by a high-voltage electric field. This method has been used for soft ionization in the mass spectrometric analysis of macromolecules and biomaterials. Moreover, in desorption electrospray ionization (DESI) for mass spectrometry, the electrically charged droplets hit the surface molecules and are ionized under ambient conditions. Electrospraying is also used for the production of nanofibers and protein chips. Delivery of a foreign nucleic acid into a living cell is an important technique in molecular biology and the medical field. Previously developed transfection techniques, including methods that use a cationic polymer, lipofection, a gene gun, and electroporation, have been reported. Moreover, Pui et al. used electrospraying to increase the momentum of particles in the gene-gun method. However, these techniques sometimes cause severe damage to cells, especially mammalian cells, or require complex and expensive devices. We hypothesized that liquid droplet impact by electrospraying would transport a nucleic acid into a cell by making a transient channel through mild damage on the cell surface. Herein, we describe a technique for the introduction of a gene into eukaryotic (mammalian) and prokaryotic (bacterial) cells and avian embryonic tissue by spraying water droplets produced by an electrospray device without any cytotoxic reagents. Figure 1 shows a diagram of the electrospray equipment for gene delivery. Purified water is sprayed onto the cells as a charged aerosol generated by static electricity repulsion at the tip of the tube. Only water or phosphate-buffered saline (PBS) is used in this method, and the device is a simple structure basically consisting of a tube connected to a highvoltage power supply that is a constant potentiostat with the current limited to 100 mA. A stainless-steel capillary (caliber 0.1 mm, outside diameter 0.3 mm, length 40 mm) was impressed with a high voltage, and water was supplied at a flow rate of 100–200 mLmin . In this experiment, the stainless-steel tube was impressed from 7 to 18 kV. This voltage (over 7 kV) is higher than that used for mass spectrometry (1–3 kV), as an organic solvent, which is usually used in mass analysis for reduction of surface tension, cannot be applied to gene transfection because of its cytotoxicity. The sprayed dishes were placed on an electrically grounded square-plate electrode, and the inside of the culture dish was also grounded by attaching a small piece of metal foil to the plate. Adhesive Chinese hamster ovary (CHO) cells and HeLa cells were tested as models, and almost the same results were obtained. CHO cells were cultured in minimum essential medium (a-MEM, Gibco, USA) supplemented with 10% fetal bovine serum. Cells were plated in a 35-mm culture dish (Falcon, USA) at 2.0 ? 10 cells per dish and cultured at 37 8C under 5% CO2 in air. Three days later, the cells were used for electrospraying. The culture medium was removed from the dish and an aqueous solution (100 mL) of plasmid vector pEGFP-N1 (100 mgmL 1 in water, Clonetech, USA), as green fluorescence protein (GFP) encoding DNA, was added to the dish. Water was electrosprayed onto the cells from a height of 2 cm at 10 kV, and culture mediumwas directly added to the dish. After 24 h of cultivation, GFP-positive cells were counted with a hemocytometer under a fluorescence microscope (Olympus, Japan). We used nonmoving equipment in this experiment, which resulted in a limited spraying area on the dish. We evaluated the transfection rate of isolated GFP-positive cells to total cells, which included cells located in the external region of the sprayed zone, and obtained a transfection rate of 0.05 to 1.6%. The number of cells showing fluorescence increased with an increase in applied voltage, which indicates that the Figure 1. Diagram of the electrospray equipment for gene delivery. The plasmid DNAs located around the plasma membrane are introduced into cells by collision of water microdroplets.

Recent progress in studies on the health benefits of pyrroloquinoline quinone

Pyrroloquinoline quinone (PQQ), an aromatic tricyclic o-quinone, was identified initially as a redox cofactor for bacterial dehydrogenases. Although PQQ is not biosynthesized in mammals, trace amounts of PQQ have been found in human and rat tissues because of its wide distribution in dietary sources. Importantly, nutritional studies in rodents have revealed that PQQ deficiency exhibits diverse systemic responses, including growth impairment, immune dysfunction, and abnormal reproductive performance. Although PQQ is not currently classified as a vitamin, PQQ has been implicated as an important nutrient in mammals. In recent years, PQQ has been receiving much attention owing to its physiological importance and pharmacological effects. In this article, we review the potential health benefits of PQQ with a focus on its growth-promoting activity, anti-diabetic effect, anti-oxidative action, and neuroprotective function. Additionally, we provide an update of its basic pharmacokinetics and safety information in oral ingestion. Graphical abstract Structure and redox reaction of pyrroloquinoline quinone (PQQ).

Collision of millimetre droplets induces DNA and protein transfection into cells

Nonperturbing and simple transfection methods are important for modern techniques used in biotechnology. Recently, we reported that electrospraying can be applied to DNA transfection in cell lines, bacteria, and chicken embryos. However, the transfection efficiency was only about 2%. To improve the transfection rate, physical properties of the sprayed droplets were studied in different variations of the method. We describe a highly efficient technique (30–93%) for introduction of materials such as DNA and protein into living cells by electrospraying droplets of a high conductivity liquid onto cells incubated with the material for transfection. Electric conductivity has a sizable influence on the success of transfection. In contrast, molecular weight of the transfected material, types of ions in the electrospray solution, and the osmotic pressure do not influence transfection efficiency. The physical analysis revealed that collision of cells with millimetre-sized droplets activates intracellular uptake.

Crystal structure and characterization of pyrroloquinoline quinone disodium trihydrate

BackgroundPyrroloquinoline quinone (PQQ), a tricarboxylic acid, has attracted attention as a growth factor, and its application to supplements and cosmetics is underway. The product used for these purposes is a water-soluble salt of PQQ disodium. Although in the past, PQQ disodiumpentahydrates with a high water concentration were used, currently, low hydration crystals of PQQ disodiumpentahydrates are preferred.ResultsWe prepared a crystal of PQQ disodium trihydrate in a solution of ethanol and water, studied its structure, and analyzed its properties. In the prepared crystal, the sodium atom interacted with the oxygen atom of two carboxylic acids as well as two quinones of the PQQ disodium trihydrate. In addition, the hydration water of the prepared crystal was less than that of the conventional PQQ disodium crystal. From the results of this study, it was found that the color and the near-infrared (NIR) spectrum of the prepared crystal changed depending on the water content in the dried samples.ConclusionsThe water content in the dried samples was restored to that in the trihydrate crystal by placing the samples in a humid environment. In addition, the results of X-ray diffraction (XRD) and X-ray diffraction-differential calorimetry (XRD-DSC) analyses show that the phase of the trihydrate crystal changed when the crystallization water was eliminated. The dried crystal has two crystalline forms that are restored to the original trihydrate crystals in 20% relative humidity (RH). This crystalline (PQQ disodium trihydrate) is stable under normal environment.

Kinetic Study of Aroxyl Radical Scavenging and α-Tocopheroxyl Regeneration Rates of Pyrroloquinolinequinol (PQQH2, a Reduced Form of Pyrroloquinolinequinone) in Dimethyl Sulfoxide Solution: Finding of Synergistic Effect on the Reaction Rate due to the Coexistence of α-Tocopherol and PQQH2

Measurements of aroxyl radical (ArO•)-scavenging rate constants (ks AOH) of antioxidants (AOHs: pyrroloquinolinequinol (PQQH2), α-tocopherol (α-TocH), ubiquinol-10 (UQ10H2), epicatechin, epigallocatechin, epigallocatechin gallate, and caffeic acid) were performed in dimethyl sulfoxide (DMSO) solution, using stopped-flow spectrophotometry. The ks AOH values were measured not only for each AOH but also for the mixtures of two AOHs ((i) α-TocH and PQQH2 and (ii) α-TocH and UQ10H2). A notable synergistic effect that the ks AOH values increase 1.72, 2.42, and 2.50 times for α-TocH, PQQH2, and UQ10H2, respectively, was observed for the solutions including two kinds of AOHs. Measurements of the regeneration rates of α-tocopheroxyl radical (α-Toc•) to α-TocH by PQQH2 and UQ10H2 were performed in DMSO, using double-mixing stopped-flow spectrophotometry. Second-order rate constants (kr) obtained for PQQH2 and UQ10H2 were 1.08 × 105 and 3.57 × 104 M−1 s−1, respectively, indicating that the kr value of PQQH2 is 3.0 times larger than that of UQ10H2. It has been clarified that PQQH2 and UQ10H2 having two HO groups within a molecule may rapidly regenerate two molecules of α-Toc• to α-TocH. The result indicates that the prooxidant effect of α-Toc• is suppressed by the coexistence of PQQH2 or UQ10H2.

Lifespan extension by peroxidase and dual oxidase-mediated ROS signaling through pyrroloquinoline quinone in C. elegans

ABSTRACT Reactive oxygen species (ROS), originally characterized based on their harmful effects on cells or organisms, are now recognized as important signal molecules regulating various biological processes. In particular, low levels of ROS released from mitochondria extend lifespan. Here, we identified a novel mechanism of generating appropriate levels of ROS at the plasma membrane through a peroxidase and dual oxidase (DUOX) system, which could extend lifespan in Caenorhabditis elegans. A redox co-factor, pyrroloquinoline quinone (PQQ), activates the C. elegans DUOX protein BLI-3 to produce the ROS H2O2 at the plasma membrane, which is subsequently degraded by peroxidase (MLT-7), eventually ensuring adequate levels of ROS. These ROS signals are transduced mainly by the oxidative stress transcriptional factors SKN-1 (Nrf2 or NFE2L2 in mammals) and JUN-1, and partially by DAF-16 (a FOXO protein homolog). Cell biology experiments demonstrated a similarity between the mechanisms of PQQ-induced activation of human DUOX1 and DUOX2 and that of C. elegans BLI-3, suggesting that DUOXs are potential targets of intervention for lifespan extension. We propose that low levels of ROS, fine-tuned by the peroxidase and dual oxidase system at the plasma membrane, act as second messengers to extend lifespan by the effect of hormesis. Summary: A peroxidase and dual oxidase system generates longevity-promoting ROS at the plasma membrane in C. elegans. Pyrroloquinoline quinone can activate the dual oxidase involved in lifespan extension.

Ultrasensitive determination of pyrroloquinoline quinone in human plasma by HPLC with chemiluminescence detection using the redox cycle of quinone

Graphical abstract Figure. No Caption available. HighlightsHPLC‐CL method was developed for PQQ analysis using the redox cycle of quinone.The method was ultrasensitive to PQQ with LOD of 1.08 nmol/L plasma (0.27 nM).A novel solid phase extraction (SPE) method was developed for PQQ from human plasma.The developed SPE method was simple, fast, and efficient (recovery >95%).The method was applied to monitor PQQ in human plasma after administration of PQQ. Abstract A fast, accurate, and ultrasensitive high‐performance liquid chromatography method with chemiluminescence detection (HPLC‐CL) was optimized and validated for the determination of pyrroloquinoline quinone (PQQ) concentration in human plasma following solid‐phase extraction (SPE). This method is based on the redox cycle of the reaction between PQQ and dithiothreitol, which generates reactive oxygen species that can be detected using luminol as a CL probe. The isocratic HPLC system comprised an ODS column and 4.0 mM tetra‐n‐butylammonium bromide in Tris‐HNO3 buffer (pH 8.8; 50 mM)‐acetonitrile (7:3, v/v) as mobile phase. A novel, rapid, and simple SPE method was also developed providing excellent %recovery (≥95.2%) for PQQ from human plasma samples. The proposed method was linear over the range of 4.0–400 nmol/L plasma of PQQ with a lower detection limit (S/N=3) of 1.08 nmol/L plasma (0.27 nM). The method was successfully implemented to determine PQQ concentration in the plasma of healthy individuals after administration of PQQ supplements.

Pyrroloquinoline Quinone, a Redox-Active o-Quinone, Stimulates Mitochondrial Biogenesis by Activating the SIRT1/PGC-1α Signaling Pathway

Pyrroloquinoline quinone (PQQ), a redox-active o-quinone found in various foods and mammalian tissues, has received an increasing amount of attention because of a number of health benefits that can be attributed to its ability to enhance mitochondrial biogenesis. However, its underlying molecular mechanism remains incompletely understood. We have now established that the exposure of mouse NIH/3T3 fibroblasts to a physiologically relevant concentration of PQQ significantly stimulates mitochondrial biogenesis. The exposure of NIH/3T3 cells to 10-100 nM PQQ for 48 h resulted in increased levels of Mitotracker staining, mitochondrial DNA content, and mitochondrially encoded cytochrome c oxidase subunit 1 (MTCO1) protein. Moreover, we observed that PQQ treatment induces deacetylation of the peroxisome proliferator-activated receptor-γ-coactivator 1α (PGC-1α) and facilitates its nuclear translocation and target gene expression but does not affect its protein levels, implying increased activity of the NAD+-dependent protein deacetylase sirtuin 1 (SIRT1). Indeed, treatment with a SIRT1 selective inhibitor, EX-527, hampered the ability of PQQ to stimulate PGC-1α-mediated mitochondrial biogenesis. We also found that the PQQ treatment caused a concentration-dependent increase in the cellular NAD+ levels, but not the total NAD+ and NADH levels. Our results suggest that PQQ-inducible mitochondrial biogenesis can be attributed to activation of the SIRT1/PGC-1α signaling pathway by enhancing cellular NAD+ formation.

Pyrroloquinoline quinone (PQQ) is reduced to pyrroloquinoline quinol (PQQH2) by vitamin C, and PQQH2 produced is recycled to PQQ by air oxidation in buffer solution at pH 7.4

Measurements of the reaction of sodium salt of pyrroloquinoline quinone (PQQNa2) with vitamin C (Vit C) were performed in phosphate-buffered solution (pH 7.4) at 25 °C under nitrogen atmosphere, using UV–vis spectrophotometry. The absorption spectrum of PQQNa2 decreased in intensity due to the reaction with Vit C and was changed to that of pyrroloquinoline quinol (PQQH2, a reduced form of PQQ). One molecule of PQQ was reduced by two molecules of Vit C producing a molecule of PQQH2 in the buffer solution. PQQH2, thus produced, was recycled to PQQ due to air oxidation. PQQ and Vit C coexist in many biological systems, such as vegetables, fruits, as well as in human tissues. The results obtained suggest that PQQ is reduced by Vit C and functions as an antioxidant in biological systems, because it has been reported that PQQH2 shows very high free-radical scavenging and singlet-oxygen quenching activities in buffer solutions. Graphical abstract Recycling of PQQH2 and PQQ under the coexistence of Vit C.

Synthesis and crystal structure of pyrroloquinoline quinol (PQQH2) and pyrroloquinoline quinone (PQQ)

Pyrroloquinoline quinone (PQQ) is a water-soluble quinone compound first identified as a cofactor of alcohol- and glucose-dehydrogenases (ADH and GDH) in bacteria. For example, in the process of ADH reaction, alcohol is oxidized to the corresponding aldehyde, and inversely PQQ is reduced to pyrroloquinoline quinol (PQQH2). PQQ and PQQH2 molecules play an important role as a cofactor in ADH and GDH reactions. However, crystal structure analysis has not been performed for PQQ and PQQH2. In the present study, the synthesis of PQQH2 powder crystals was performed under air, by utilizing vitamin C as a reducing agent. By reacting a trihydrate of disodium salt of PQQ (PQQNa2·3H2O) with excess vitamin C in H2O at 293 and 343 K, yellowish brown and black powder crystals of PQQH2 having different properties were obtained in high yield, respectively. The former was PQQH2 trihydrate (PQQH2·3H2O) and the latter was PQQH2 anhydrate (PQQH2). Furthermore, sodium-free red PQQ powder crystal (a monohydrate of PQQ, PQQ·H2O) was prepared by the reaction of PQQNa2·3H2O with HCl in H2O. Single crystals of PQQH2 and PQQ were prepared from Me2SO/CH3CN mixed solvent, and we have succeeded in the crystal structure analyses of PQQH2 and PQQ for the first time.