Masato Yasuhara

Quantum Dots Targeted to the Assigned Organelle in Living Cells

Fluorescent nanocrystal quantum dots (QDs) have the potential to be applied to bioimaging since QDs emit higher and far longer fluorescence than conventional organic probes. Here we show that QDs conjugated with signal peptide obey the order to transport the assigned organelle in living cells. We designed the supermolecule of luminescent QDs conjugated with nuclear‐ and mitochondria‐targeting ligands. When QDs with nuclear‐localizing signal peptides were added to the culture media, we can visualize the movements of the QDs being delivered into the nuclear compartment of the cells with 15 min incubation. In addition, mitochondrial signal peptide can also transport QDs to the mitochondria in living cells. In conclusion, these techniques have the possibility that QDs can reveal the transduction of proteins and peptides into specific subcellular compartments as a powerful tool for studying intracellular analysis in vitro and even in vivo.

Use of fluorescent quantum dot bioconjugates for cellular imaging of immune cells, cell organelle labeling, and nanomedicine: surface modification regulates biological function, including cytotoxicity.

With the development of nanotechnology, nanoscale products that are smaller than several hundred nanometers have been applied to all areas of science and technology. Nanoscale products, including carbon nanotubes, fullerene derivatives, and nanocrystal quantum dots (QDs), are wide spread as novel tools in various fields, not only in materials engineering, electronics, plastics, and the automobile and aerospace industries, but also in molecular biology and medicine. At present, QDs have been widely used in biological and medical studies because of their superior photoemission and photostability. Although the physical and chemical properties of QDs have been circumstantially investigated, little is known about any harmful effects of QDs on human health. Here we report on the toxicity and biological behavior of QDs in vitro and in vivo. The toxicity of the core constituent chemicals such as cadmium and selenium has been identified. Recently, the surface molecules surrounding QDs have been intensively investigated. Accumulating evidence that toxic surface-covering molecules showed their cytotoxicity and biomolecules conjugated with QDs maintained their biological effects indicates that at least the biological properties of QDs are attributable to the QD-capping material rather than to the core metalloid complex itself.

Inhibition of CCL1-CCR8 Interaction Prevents Aggregation of Macrophages and Development of Peritoneal Adhesions

Peritoneal adhesions are a significant complication of surgery and visceral inflammation; however, the mechanism has not been fully elucidated. The aim of this study was to clarify the mechanism of peritoneal adhesions by focusing on the cell trafficking and immune system in the peritoneal cavity. We investigated the specific recruitment of peritoneal macrophages (PMφ) and their expression of chemokine receptors in murine models of postoperative and postinflammatory peritoneal adhesions. PMφ aggregated at the site of injured peritoneum in these murine models of peritoneal adhesions. The chemokine receptor CCR8 was up-regulated in the aggregating PMφ when compared with naive PMφ. The up-regulation of CCR8 was also observed in PMφ, but not in bone marrow-derived Mφ, treated with inflammatory stimulants including bacterial components and cytokines. Importantly, CCL1, the ligand for CCR8, a product of both PMφ and peritoneal mesothelial cells (PMCs) following inflammatory stimulation, was a potent enhancer of CCR8 expression. Cell aggregation involving PMφ and PMCs was induced in vitro in the presence of CCL1. CCL1 also up-regulated mRNA levels of plasminogen activator inhibitor-1 in both PMφ and PMCs. CCR8 gene-deficient mice or mice treated with anti-CCL1-neutralizing Ab exhibited significantly reduced postoperational peritoneal adhesion. Our study now establishes a unique autocrine activation system in PMφ and the mechanism for recruitment of PMφ together with PMCs via CCL1/CCR8, as immune responses of peritoneal cavity, which triggers peritoneal adhesions.

Deficiency of Chemokine Receptor CCR1 Causes Osteopenia Due to Impaired Functions of Osteoclasts and Osteoblasts

Chemokines are characterized by the homing activity of leukocytes to targeted inflammation sites. Recent research indicates that chemokines play more divergent roles in various phases of pathogenesis as well as immune reactions. The chemokine receptor, CCR1, and its ligands are thought to be involved in inflammatory bone destruction, but their physiological roles in the bone metabolism in vivo have not yet been elucidated. In the present study, we investigated the roles of CCR1 in bone metabolism using CCR1-deficient mice. Ccr1−/− mice have fewer and thinner trabecular bones and low mineral bone density in cancellous bones. The lack of CCR1 affects the differentiation and function of osteoblasts. Runx2, Atf4, Osteopontin, and Osteonectin were significantly up-regulated in Ccr1−/− mice despite sustained expression of Osterix and reduced expression of Osteocalcin, suggesting a lower potential for differentiation into mature osteoblasts. In addition, mineralized nodule formation was markedly disrupted in cultured osteoblastic cells isolated from Ccr1−/− mice. Osteoclastogenesis induced from cultured Ccr1−/− bone marrow cells yielded fewer and smaller osteoclasts due to the abrogated cell-fusion. Ccr1−/− osteoclasts exerted no osteolytic activity concomitant with reduced expressions of Rank and its downstream targets, implying that the defective osteoclastogenesis is involved in the bone phenotype in Ccr1−/− mice. The co-culture of wild-type osteoclast precursors with Ccr1−/− osteoblasts failed to facilitate osteoclastogenesis. This finding is most likely due to a reduction in Rankl expression. These observations suggest that the axis of CCR1 and its ligands are likely to be involved in cross-talk between osteoclasts and osteoblasts by modulating the RANK-RANKL-mediated interaction.

Simultaneous multicolor detection system of the single-molecular microbial antigen with total internal reflection fluorescence microscopy.

Immunological diagnostic methods have been widely performed and showed high performance in molecular and cellular biology, molecular imaging, and medical diagnostics. We have developed novel methods for the fluorescent labeling of several antibodies coupled with fluorescent nanocrystal QDs. In this study we demonstrated that two bacterial toxins, diphtheria toxin and tetanus toxin, were detected simultaneously in the same view field of a cover slip by using directly QD‐conjugated antibodies. We have succeeded in detecting bacterial toxins by counting luminescent spots on the evanescent field with using primary antibody conjugated to QDs. In addition, each bacterial toxin in the mixture can be separately detected by single excitation laser with emission band pass filters, and simultaneously in situ pathogen quantification was performed by calculating the luminescent density on the surface of the cover slip. Our results demonstrate that total internal reflection fluorescence microscopy (TIRFM) enables us to distinguish each antigen from mixed samples and can simultaneously quantitate multiple antigens by QD‐conjugated antibodies. Bioconjugated QDs could have great potentialities for in practical biomedical applications to develop various high‐sensitivity detection systems.

Separation of murine neutrophils and macrophages by thermoresponsive magnetic nanoparticles.

Magnetic particles have been used widely in both biotechnological and medical fields, including for immunoassay, enzyme immobilization, drug transport, and immunological diagnosis. Especially particles with bioactive molecules such as antibodies and streptavidin are very useful tools for cell separation. Here we report affinity selection of neutrophils and macrophages from peritoneal inflammatory cells performed by thermoresponsive magnetic nanoparticles conjugated with macrophage‐specific anti‐F4/80 antibody. The magnetic nanoparticles, which are capped with thermoresponsive polymers, are aggregated by heating the particles over 30 °C and show their intrinsic magnetism. The neutrophils are concentrated approximately 90% by these magnetic nanoparticles without any activation, indicating that this novel cell separation method could fulfill a wide range of applications in analysis of the isolation of fragile cells such as neutrophils.

GFP expression by intracellular gene delivery of GFP-coding fragments using nanocrystal quantum dots

Gene therapy is an attractive approach to supplement a deficient gene function. Although there has been some success with specific gene delivery using various methods including viral vectors and liposomes, most of these methods have a limited efficiency or also carry a risk for oncogenesis. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully introduced gene-fragments with promoter elements, which promoted the expression of the enhanced green fluorescent protein (eGFP) gene in mammalian cells. The expression of eGFP protein was observed when the QD/gene-construct was added to the culture media. The gene-expression efficiency varied depending on multiple factors around QDs, such as (1) the reading direction of the gene-fragments, (2) the quantity of gene-fragments attached on the surface of the QD-constructs, (3) the surface electronic charges varied according to the structure of the QD/gene-constructs, and (4) the particle size of QD/gene complex varied according to the structure and amounts of gene-fragments. Using this QD/gene-construct system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs had disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without using either viral vectors or specific liposomes.

The increased intestinal absorption rate is responsible for the reduced hepatic first‐pass extraction of propranolol in rats with cisplatin‐induced renal dysfunction

The mechanisms responsible for the increased bioavailability of propranolol in renal dysfunction were investigated in rats. Experimental acute renal failure (ARF) was induced by intraperitoneal injection of cisplatin (5 mg kg−1). ARF induced a significant increase in blood propranolol concentration after intra‐intestinal administration. The extent of bioavailability (F) of propranolol at an intestinal dose of 15 mg kg−1 was 16.4% and 26.9% in control and ARF rats, respectively, and the F value at a 37.5 mg kg−1 dose was 54.7% and 81.4% in control and ARF rats, respectively. In contrast, the blood propranolol concentration following intraportal infusion was not increased significantly in ARF rats. The hepatic first‐pass extraction (Eh) was dose‐dependent and saturable: Eh of propranolol in control rats was 58.0% and 18.3% at 8 and 20 mg kg−1, respectively, and Eh in ARF rats was 50.8% and 19.9% at 8 and 20 mg kg−1, respectively. The initial absorption rate of propranolol from the intestine in ARF rats was significantly greater compared with control rats. These results indicated that the increased bioavailability of propranolol in rats with cisplatin‐induced renal dysfunction was mainly a result of the increased absorption rate in the intestine followed by the partial saturation of hepatic first‐pass metabolism.

Trafficking of QD-Conjugated MPO-ANCA in Murine Systemic Vasculitis and Glomerulonephritis Model Mice

In systemic vasculitis, the serum level of myeloperoxidase (MPO)‐specific anti‐neutrophil cytoplasmic autoantibodies (MPO‐ANCA) is significantly elevated with the progression of disease. We have established a model of murine systemic vasculitis by administration of MPO‐ANCA and fungal mannoprotein to C57BL/6 mice. We examined the role of MPO and MPO‐ANCA in the pathogenesis of glomerulonephritis and systemic vasculitis in this model using quantum dots (QDs). We demonstrated that QD‐conjugated MPO‐ANCA (ANCA‐QD) visualized the translocation of MPO on the neutrophil membrane surface after stimulation with proinflammatory cytokines. We also observed that MPO translocation on neutrophils in both patients with rapid progressive glomerulonephritis and these model mice without any stimulation, suggesting that MPO translocation is certain to contribute to the development of glomerular lesion. In addition, blood flow on the kidney surface vessel was significantly decelerated in both SCG/Kj mice and this model, suggesting that ANCA induces the damage of blood vessel. These results indicate that MPO‐ANCA and surface‐translocated MPO on the activated neutrophils coordinately plays essential roles in the initial steps of the glomerulonephritis.

Effect of experimental renal dysfunction on bioavailability of ajmaline in rats

The effect of renal dysfunction on the bioavailability of ajmaline has been investigated in rats, where experimental renal dysfunction was induced by subcutaneous injection of uranyl nitrate (10 mg kg−1). Renal dysfunction did not cause any change in the blood ajmaline concentration after intravenous administration (2 mg kg−1), but it increased the blood ajmaline concentration by approximately 2.8‐fold after intraduodenal administration (10 mg kg−1). The availability of ajmaline in control rats was 16.7%, whereas the availability was increased to 41.1% in rats with renal dysfunction. The unbound fraction in the blood and the metabolic activity in the liver, was assessed with the 10000‐g supernatant fraction and with isolated hepatocytes, respectively. The values were found to be similar in both groups. The blood concentration following intraportal infusion was only slightly increased in rats with renal dysfunction, but the hepatic first‐pass extraction was infusion rate‐dependent and saturable. The initial absorption rate of ajmaline from the small intestine in rats with renal dysfunction was significantly greater compared with control rats. These results indicated that the increased availability of ajmaline in renal dysfunction was mainly a result of partially saturated extraction in the liver, which was caused by an increased absorption rate in the intestine and non‐linear extraction in the liver.