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Suzuki M.,Waseda Bioscience Research Institute in Singapore | Suzuki M.,Waseda University | Ishiwata S.,Waseda Bioscience Research Institute in Singapore | Ishiwata S.,Waseda University
Biophysical Journal | Year: 2011

Electron microscopy has shown that cross-bridges (CBs) are formed at the target zone that is periodically distributed on the thin filament in striated muscle. Here, by manipulating a single bead-tailed actin filament with optical tweezers, we measured the unbinding events of rigor CBs one by one on the surface of the A-band in rabbit skeletal myofibrils. We found that the spacings between adjacent CBs were not always the same, and instead were 36, 72, or 108 nm. Tropomyosin and troponin did not affect the CB spacing except for a relative increase in the appearance of longer spacing in the presence of Ca 2+. In addition, in an in vitro assay where myosin molecules were randomly distributed, were obtained the same spacing, i.e., a multiple of 36 nm. These results indicate that the one-dimensional distribution of CBs matches with the 36-nm half pitch of a long helical structure of actin filaments. A stereospecific model composed of three actin protomers per target zone was shown to explain the experimental results. Additionally, the unbinding force (i.e., the binding affinity) of CBs for the reconstituted thin filaments was found to be larger and smaller relative to that for actin filaments with and without Ca 2+, respectively. © 2011 Biophysical Society.


Sakai H.,Waseda University | Sakai H.,Waseda Bioscience Research Institute in Singapore | Suzuki Y.,Nihon Bioresearch Inc. | Sou K.,Waseda University | Kano M.,Nihon Bioresearch Inc.
Journal of Biomedical Materials Research - Part A | Year: 2012

Intravenous injection of liposomes into pigs reportedly induces anaphylactoid reactions at a small dose, resulting in circulatory disorder. Hemoglobin vesicles (HbVs) are artificial oxygen carriers encapsulating Hb solution in liposomes. It is not known how pigs respond to HbV injection. We aimed to analyze the cardiopulmonary responses to small injections of HbV and empty vesicle (EV) and compare them with a conventional liposome (CL) with a different lipid composition containing phosphatidylglycerol (PG). PG is known to induce an anaphylactoid reaction in pigs. Nine male miniature pigs were used for HbV, EV, and CL injections. The anesthetized pig received 0.05 and 0.5 mL/kg of a test fluid for the first and second injection with a 70 min interval. Results show that CL repeatedly induced significant increases in systemic and pulmonary arterial pressures and vascular resistances and decreases in heart rate and cardiac output (CO). HbV and EV at the first injection-induced pulmonary hypertension, with significantly smaller changes in systemic arterial pressure and CO. No remarkable response was visible at the second injection in spite of a larger dosage. Only CL repeatedly induced thrombocytopenia, leukocytopenia, and plasma thromboxane B2 increase resulting from complement activation, although HbV and EV showed smaller changes. Transmittance electron micrograph of pulmonary intravascular macrophages (PIMs) showed phagocytosis of HbV, indicating the possibility that nonspecific phagocytosis by PIMs relates to the responses observed after the first injection. HbV does not induce a significant anaphylactoid reaction in pigs compared with CL because of the different lipid composition. © 2012 Wiley Periodicals, Inc.


Sakai H.,Nara Medical University | Sakai H.,Waseda Bioscience Research Institute in Singapore | Sakai H.,Waseda University | Li B.,Waseda Bioscience Research Institute in Singapore | And 2 more authors.
Bioconjugate Chemistry | Year: 2014

Electron-energy-rich coenzymes in cells, NADH and NADPH, are re-energized repeatedly through the Embden-Meyerhof and pentose-phosphate glycolytic pathways, respectively. This study demonstrates extraction of their electron energies in red blood cells (RBCs) for in vivo extracellular chemical reactions using an electron mediator shuttling across the biomembrane. Hemoglobin-vesicles (HbVs) are an artificial oxygen carrier encapsulating purified and concentrated Hb solution in liposomes. Because of the absence of a metHb-reducing enzymatic system in HbV, HbO2 gradually autoxidizes to form metHb. Wistar rats received HbV suspension (10 mL/kg body weight) intravenously. At the metHb level of around 50%, methylene blue [MB+; 3,7-bis(dimethylamino) phenothiazinium chloride] was injected. The level of metHb quickly decreased to around 16% in 40 min, remaining for more than 5 h. In vitro mixing of HbV/MB+ with RBCs recreated the in vivo metHb reduction, but not with plasma. NAD(P)H levels in RBCs decreased after metHb reduction. The addition of glucose facilitated metHb reduction. Liposome-encapsulated NAD(P)H, a model of RBC, reduced metHb in HbV in the presence of MB+. These results indicate that (i) NAD(P)H in RBCs reacts with MB+ to convert it to leukomethylene blue (MBH); (ii) MB+ and MBH shuttle freely between RBC and HbV across the hydrophobic lipid membranes; and (iii) MBH is transferred into HbV and reduces metHb in HbV. Four other electron mediators with appropriate redox potentials appeared to be as effective as MB+ was, indicating the possibility for further optimization of electron mediators. We established an indirect enzymatic metHb reducing system for HbV using unlimited endogenous electrons created in RBCs in combination with an effective electron mediator that prolongs the functional lifespan of HbV in blood circulation. © 2014 American Chemical Society.


Fujihara M.,Red Cross | Takahashi D.,Red Cross | Abe H.,Red Cross | Sakai H.,Waseda Bioscience Research Institute in Singapore | And 4 more authors.
Artificial Organs | Year: 2014

Hemoglobin vesicles (HbVs), artificial oxygen carriers encapsulating concentrated Hb solution on phospholipid vesicles (liposomes), are promising candidates for clinically useful transfusion. Although HbV infusion transiently suppressed the proliferative response of rat splenic T-cells to concanavalin A or keyhole limpet hemocyanin (KLH), a T-cell-dependent antigen, in ex vivo culture conditions, HbV infusion did not affect the primary IgG antibody response. We extended our assessment of the effects of HbV infusion on the systemic immune response using primary and secondary responses to KLH in rats. We observed that the generation of primary anti-KLH IgM antibody in HbV-infused rats was not suppressed but was instead higher than those in saline-infused rats. Furthermore, HbV infusion did not suppress the increase of IgG subclass of KLH antibody in secondary response. The T cell response to KLH of bulk spleen cells, as derived from 2-3 months after secondary KLH immunization, was unaffected by infusion of HbV, suggesting that HbV loading has no suppressive effect on homeostatic survival of memory T-cells against KLH. These results indicate that HbV is highly biocompatible in systemic immune responses in rats. © 2013 Wiley Periodicals, Inc. and International Center for Artificial Organs and Transplantation.


Sakai H.,Waseda University | Sakai H.,Waseda Bioscience Research Institute in Singapore | Okuda N.,Waseda University | Sato A.,Waseda University | And 4 more authors.
American Journal of Physiology - Heart and Circulatory Physiology | Year: 2010

Intravenous administration of cell-free Hb induces vasoconstriction and circulatory disorders, presumably because of the intrinsic affinities to endogenous nitric oxide (NO) and carbon monoxide (CO) as vasorelaxation factors and because of the facilitated O2 release that might induce autoregulatory vasoconstriction. We examined these gas reactions when Hb-containing solutions of four kinds were perfused through artificial narrow tubes at a practical Hb concentration (10 g/dl). Purified Hb solution, polymerized bovine Hb (PolyBHb), encapsulated Hb [Hb-vesicles (HbV), 279 nm], and red blood cells (RBCs) were perfused through a gas-permeable narrow tube (25 μm inner diameter) at 1 mm/s centerline velocity. The level of reactions was determined microscopically based on the visible-light absorption spectrum of Hb. When the tube was immersed in NO and CO atmospheres, both NO binding and CO binding of deoxygenated Hb (deoxy-Hb) and PolyBHb in the tube was faster than those of HbV and RBCs, and HbV and RBCs showed almost identical binding rates. When the tube was immersed in a N2 atmosphere, oxygenated Hb and PolyBHb showed much faster O2 release than did HbV and RBCs. PolyBHb showed a faster reaction than Hb because of the lower O2 affinity of PolyBHb than Hb. The diffusion process of the particles was simulated using Navier-Stokes and Maxwell-Stefan equations. Results clarified that small Hb (6 nm) diffuses laterally and mixes rapidly. However, the large-dimension HbV shows no such rapid diffusion. The purely physicochemical differences in diffusivity of the particles and the resulting reactivity with gas molecules are one factor inducing biological vasoconstriction of Hb-based oxygen carriers. Copyright © 2010 American Physiological Society.


Ong P.K.,National University of Singapore | Jain S.,National University of Singapore | Namgung B.,National University of Singapore | Woo Y.I.,National University of Singapore | And 4 more authors.
Physiological Measurement | Year: 2011

Histogram-based thresholding techniques utilized for cell-free layer width measurement in arteriolar flow may produce an overestimation of the layer width since they do not consider faint shaded regions near the vessel wall as part of the erythrocyte column. To address this problem, we developed a new method for detecting the boundary of the erythrocyte column based on an edge detection algorithm. This automated method (grayscale method) provides local detections of the inner vessel wall as well as the boundary between the cell-free layer and the erythrocyte column without binarization of grayscale images. The cell-free layer width measurements using the grayscale method and existing techniques (minimum method and Otsu's method) were compared with those determined manually in arteriolar flows of the rat cremaster muscle. In the absence of the shaded regions, values obtained by the grayscale method and minimum method were statistically in good agreement with the manual method but not in the case of Otsu's method. When the faint shaded regions were present, the grayscale method appeared to produce more accurate results than the minimum method and Otsu's method. © 2011 Institute of Physics and Engineering in Medicine.


Sakai H.,Waseda Bioscience Research Institute in Singapore | Sakai H.,Waseda University | Sou K.,Waseda University | Horinouchi H.,Keio University | And 2 more authors.
Artificial Organs | Year: 2012

The hemoglobin-vesicle (HbV) is an artificial oxygen carrier encapsulating a concentrated hemoglobin solution in a phospholipid vesicle (liposome). During or after transporting oxygen, macrophages capture HbVs in the reticuloendothelial system (RES) with an approximate circulation half-life of 3 days. Animal studies show transient splenohepatomegaly after large doses, but HbVs were completely degraded, and the components were excreted in a few weeks. If a blood substitute is used for emergency use until red blood cell transfusion becomes available or for temporary use such as a priming fluid for an extracorporeal circuit, then one option would be to remove HbVs from the circulating blood without waiting a few weeks for removal by the RES. Using a mixture of beagle dog whole blood and HbV, we tested the separation of HbV using a centrifugal Fresenius cell separator and an ultrafiltration system. The cell separator system separated the layers of blood cell components from the HbV-containing plasma layer by centrifugal force, and then the HbV was removed from plasma phase by the ultrafiltration system. The HbVs (250-280nm) are larger than plasma proteins (<22nm diameter) but smaller than blood cell components (>3μm). The size of HbVs is advantageous to be separated from the original blood components, and the separated blood components can be returned to circulation. © 2011, Copyright the Authors. Artificial Organs © 2011, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.


Shimomura T.,Waseda University | Iwamoto H.,Japan Synchrotron Radiation Research Institute | Vo Doan T.T.,Nanyang Technological University | Ishiwata S.,Waseda University | And 4 more authors.
Biophysical Journal | Year: 2016

In contrast to major flight muscles in the Mecynorrhina torquata beetle, the third axillary (3Ax) muscle is a minor flight muscle that uniquely displays a powerful mechanical function despite its considerably small volume, ∼1/50 that of a major flight muscle. The 3Ax muscle contracts relatively slowly, and in flight strongly pulls the beating wing to attenuate the stroke amplitude. This attenuation leads to left-right turning in flight or wing folding to cease flying. What enables this small muscle to be so powerful? To explore this question, we examined the microstructure of the 3Ax muscle using synchrotron x-ray diffraction, optical microscopy, and immunoblotting analysis. We found that the 3Ax muscle has long (∼5 μm) myofilaments and that the ratio of thick (myosin) filaments to thin (actin) filaments is 1:5 or 1:6. These characteristics are not observed in the major flight muscles, which have shorter myofilaments (∼3.5 μm) with a smaller ratio (1:3), and instead are more typical of a leg muscle. Furthermore, the flight-muscle-specific troponin isoform, TnH, is not expressed in the 3Ax muscle. Since such a microstructure is suitable for generating large tension, the 3Ax muscle is appropriately designed to pull the wing strongly despite its small volume. © 2016 Biophysical Society


Takahashi D.,Red Cross | Azuma H.,Red Cross | Azuma H.,Hokkaido University | Sakai H.,Waseda Bioscience Research Institute in Singapore | And 9 more authors.
Journal of Pharmacology and Experimental Therapeutics | Year: 2011

Liposomes reportedly accumulate in monophagocytic systems (MPSs), such as those of the spleen. Accumulation of considerable amounts of liposome in a MPS can affect immunologic response. While developing a liposomal oxygen carrier containing human hemoglobin vesicle (HbV), we identified its suppressive effect on the proliferation of rat splenic T cells. The aim of this study was to elucidate the mechanism underlying that phenomenon and its effect on both local and systemic immune response. For this study, we infused HbV intravenously at a volume of 20% of whole blood or empty liposomes into rats, removed their spleens, and evaluated T cell responses to concanavalin A (Con A) or keyhole limpet hemocyanin (KLH) by measuring the amount of [3H]thymidine incorporated into DNA. Cells that phagocytized liposomal particles were sorted using flow cytometry and analyzed. Serum anti-KLH antibody was measured after immunizing rats with KLH. Results showed that T cell proliferation in response to Con A or KLH was inhibited from 6 h to 3 days after the liposome injection. Direct cell-to-cell contact was necessary for the suppression. Both inducible nitric-oxide synthase and arginase inhibitors restored T cell proliferation to some degree. The suppression abated 7 days later. Cells that trapped vesicles were responsible for the suppression. Most expressed CD11b/c but lacked class II molecules. However, the primary antibody response to KLH was unaffected. We conclude that the phagocytosis of the large load of liposomal particles by rat CD11b/c+, class II immature monocytes temporarily renders them highly immunosuppressive, but the systemic immune response was unaffected. Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics.


PubMed | Waseda Bioscience Research Institute in Singapore
Type: Journal Article | Journal: Artificial organs | Year: 2012

The hemoglobin-vesicle (HbV) is an artificial oxygen carrier encapsulating a concentrated hemoglobin solution in a phospholipid vesicle (liposome). During or after transporting oxygen, macrophages capture HbVs in the reticuloendothelial system (RES) with an approximate circulation half-life of 3 days. Animal studies show transient splenohepatomegaly after large doses, but HbVs were completely degraded, and the components were excreted in a few weeks. If a blood substitute is used for emergency use until red blood cell transfusion becomes available or for temporary use such as a priming fluid for an extracorporeal circuit, then one option would be to remove HbVs from the circulating blood without waiting a few weeks for removal by the RES. Using a mixture of beagle dog whole blood and HbV, we tested the separation of HbV using a centrifugal Fresenius cell separator and an ultrafiltration system. The cell separator system separated the layers of blood cell components from the HbV-containing plasma layer by centrifugal force, and then the HbV was removed from plasma phase by the ultrafiltration system. The HbVs (250-280 nm) are larger than plasma proteins (< 22 nm diameter) but smaller than blood cell components (> 3 m). The size of HbVs is advantageous to be separated from the original blood components, and the separated blood components can be returned to circulation.

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