Propulsive forces on water polo players' feet from eggbeater kicking estimated by pressure distribution analysis.

The purpose of this study was to characterise the unsteady propulsive force during eggbeater kicking by a fluid force estimation method based on pressure distribution analysis. The eggbeater kick was performed by six male water polo players. The participants' eggbeater kicking motions were recorded by three cameras, and the kinematic foot variables were analysed. The pressure distributions around the foot were measured by four pairs of pressure sensors attached to the dorsal and plantar surfaces of the participants' right foot. The resultant fluid force acting on the foot was estimated from the measured pressure and area of the foot. The calculated propulsive force increased with the pressure difference between the plantar and dorsal sides of the foot, which was mainly related to the decrease in pressure on the dorsal side, and peaked when the foot passed its maximum velocity and began to decelerate. These results cannot be elucidated only by conventional biomechanical theories of swimming propulsion (Newton's laws of motion and the quasi-steady approach) but instead indicate a high possibility that the exerted propulsive force is induced by the effects of unsteady water flow.

Effects of muscle cooling on kinetics of pulmonary oxygen uptake and muscle deoxygenation at the onset of exercise.

This study investigated effects of skeletal muscle cooling on the metabolic response and kinetics of pulmonary oxygen uptake ( V˙ O2 ) and skeletal muscle deoxygenation during submaximal exercise. In the cooling condition (C), after immersion of the lower body into 12°C water for 30 min, eight healthy males performed 30-min cycling exercise at the lactate threshold while undergoing thigh cooling by a water-circulating pad. In the normal condition (N) as control, they conducted the same exercise protocol without cooling. Blood lactate concentration was significantly higher in C than N at 10 min after onset of exercise (4.0 ± 1.7 and 2.4 ± 1.2 mmol/L in C and N, P < 0.05). The percent change in the tissue oxygen saturation of the vastus lateralis, measured by a near-infrared spectroscopy, was significantly lower in C at 2, 8, 10, and 20 min after the exercise onset compared with N (P < 0.05). The percent change in deoxy hemoglobin+myoglobin concentration (Deoxy[Hb+Mb]) showed a transient peak at the onset of exercise and significantly higher value in C at 10, 20, and 30 min after the exercise onset (P < 0.05). Compared to N, slower V˙ O2 kinetics (mean response time) was observed in C (45.6 ± 7.8 and 36.1 ± 7.7 sec in C and N, P < 0.05). The mean response time in C relative to N was significantly correlated with the transient peak of Deoxy[Hb+Mb] in C (r = 0.84, P < 0.05). These results suggest that lower oxygen delivery to the hypothermic skeletal muscle might induce greater glycolytic metabolism during exercise and slower V˙ O2 kinetics at the onset of exercise.

Estimation of the Number of Quantum Dots Immobilized on an Ultra-flat Au Surface.

Quantum dots (QDs) were immobilized on an ultra-flat Au surface by using amide binding between the carboxyl groups on the QDs and the amino groups of the self-assembled monolayer on the surface. The number density of the QDs estimated by atomic force microscopy (AFM) agreed with the quantity of QDs estimated by X-ray photoelectron spectroscopy and fluorescence microscopy. QDs were also immobilized on dot patterns fabricated by e-beam lithography. AFM was able to identify clusters of just a few QDs on the dot patterns, whose minimum designed size was 50 nm × 50 nm per dot.

Self-Spreading of Lipid Bilayer on a Hydrophobic Surface Made by Self-Assembled Monolayer with Short Alkyl Chain.

Behaviors of self-spreading of lipid bilayer membrane on a glass surface modified with self-assembled monolayer (SAM) with short alkyl chain were observed with fluorescence microscopy. Hydrophobic surface made by SAM was found to hamper the self-spreading phenomenon but the lipid bilayer spread on a hydrophilic one where SAM was decomposed by oxidation. On a binary surface having a hydrophobic region and a hydrophilic one, the lipid bilayer spread on the hydrophilic region but it stopped at the boundary of the hydrophobic region.

Effect of high-energy neutral particles on extreme ultraviolet spectroscopy in large helical device.

Spectra measured by an extreme ultraviolet (EUV) spectrometer frequently suffer large spike noise when Large Helical Device is operated in low-density range (≤ 3 × 10(13) cm(-3)) with neutral beam injection (NBI). The spike noise completely disappears in electron cyclotron heating discharges. In order to examine the effect of NBI, a carbon filter with thickness of 150 nm was installed in the EUV spectrometer. As a result, the spike noise was reduced by an order of magnitude. It is experimentally verified that the spike noise is caused by escaping high-energy neutral particles resulting from the circulating high-energy hydrogen ions borne from NBI.

High resolution extreme ultraviolet spectrometer for an electron beam ion trap.

An extreme ultraviolet spectrometer has been developed for spectroscopic studies of highly charged ions with an electron beam ion trap. It has a slit-less configuration with a spherical varied-line-spacing grating that provides a flat focal plane for grazing incidence light. Alternative use of two different gratings enables us to cover the wavelength range 1-25 nm. Test observations with the Tokyo electron beam ion trap demonstrate the high performance of the present spectrometer such as a resolving power of above 1000.

Production of anti-carbohydrate antibodies by phage display technologies: potential impairment of cell growth as a result of endogenous expression.

Anti-mannotriose (Man3) antibodies were previously isolated from a Keio phage library displaying human single chain variable fragments (scFvs) using a neoglycolipid, Man3- dipalmitoylphosphatidylethanolamine. Of three genes constructed, the 5A3 clone was expressed in mouse myeloma NS0 cells as a conjugate with human IgG(1) Fc (scFv-Fc) and characterized (Sakai, K., Shimizu, Y., Chiba, T., Matsumoto-Takasaki, A., Kusada, Y., Zhang, W., Nakata, M., Kojima, N., Toma, K., Takayanagi, A., Shimizu, N., and Fujita-Yamaguchi, Y. (2007) Biochemistry 46, 253-262; Zhang, W., Matsumoto-Takasaki, A., Kusada, Y., Sakaue, H., Sakai, K., Nakata, M., and Fujita-Yamaguchi, Y. (2007) Biochemistry 46, 263-270). Similarly, anti-Le(x) phages were screened from the same library with lacto-N-fucopentaose III (LNFPIII; Le(x))-dipalmitoylphosphatidylethanolamine. Of five phage clones isolated, two scFv genes were constructed to express scFv-Fc proteins in NS0 cells. As was experienced with anti-Man3 scFv-Fc clones, only one anti-LNFPIII clone, 1F12, was successfully produced and purified as an scFv-Fc protein. Although anti-LNFPIII 1F12 and anti-Man3 5A3 scFv-Fc proteins were secreted into media, a decline in scFv-Fc production was observed with both stable clones during early passages. Transient expression of anti-LNFPIII and anti-Man3 scFv-Fc genes in COS-7 cells and subsequent analyses of scFv-Fc protein expression revealed accumulation of translated proteins in the endoplasmic reticulum for scFv-Fc proteins derived from clones that did not survive as stable clones. This report describes the following: (i) isolation of anti-LNFPIII scFv genes; (ii) purification of anti-LNFPIII scFv-Fc proteins from stably and transiently expressed cells; and (iii) extracellular or intracellular localization of two anti-LNFPIII and three anti-Man3 scFv-Fc proteins. The results suggest that expression of anti-Man3 and other anti-carbohydrate antibodies in mammalian cells is disadvantageous for cell growth.

Direct fabrication of nanopores in a metal foil using focused ion beam with in situ measurements of the penetrating ion beam current.

A through hole with a diameter less than 100 nm was fabricated in an Ag foil using only a focused ion beam (FIB) system and in situ measurements of the penetrating ion beam. During the drilling of the foil by a FIB of Ga(+) ions, the transmitted part of the beam was measured with an electrode mounted on the back face of the foil. When the beam current penetrating through the nanopore reached a certain value, irradiation was stopped and the area of the created aperture was measured with a scanning electron microscope. The resulting area was correlated with the current of the penetrating ion beam. This suggests that we can fabricate a nanopore of the desired size by controlling the ion beam via penetrating ion beam measurements. The smallest aperture thus created was circular with diameter of 30 nm.

Compact electron beam ion trap for spectroscopy of moderate charge state ions.

A compact electron beam ion trap (EBIT) has been constructed for spectroscopic studies of moderate charge state ions. The electron beam energy range of the present EBIT is 100-1000 eV, for which it is rather difficult to operate an ordinary EBIT which used to be designed for operation with higher electron energy (~10 keV or more). To cut down the running costs, a superconducting wire with a high critical temperature is used for the central magnet so that it can be operated without liquid helium. The performance of the compact EBIT has been investigated through visible spectroscopy of highly charged krypton and iron ions.

Evidence for strong Breit interaction in dielectronic recombination of highly charged heavy ions.

Resonant strengths have been measured for dielectronic recombination of Li-like iodine, holmium, and bismuth using an electron beam ion trap. By observing the atomic number dependence of the state-resolved resonant strength, clear experimental evidence has been obtained that the importance of the generalized Breit interaction (GBI) effect on dielectronic recombination increases as the atomic number increases. In particular, it has been shown that the GBI effect is exceptionally strong for the recombination through the resonant state [1s2s(2)2p(1/2)](1).

Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 2. Expression, purification, and characterization of recombinant single chain antibodies.

Since phage-display technology is probably the best available strategy to produce antibodies directed against various carbohydrate moieties, we employed phage-display technology to generate human single chain antibodies (scFvs) using neoglycolipids as carbohydrate antigens. An accompanying paper in this issue describes how phage-displayed antibodies (phage Abs) that recognized nonreducing terminal mannose residues were isolated and characterized. In this study, four independent scFv genes, isolated by a mannotriose (Man3)-bearing lipid as an antigen as previously described, were used to construct expression vectors to produce soluble scFv proteins in quantity. Both bacterial and mammalian expression systems were used to produce glutathione S-transferase-scFv fusion proteins and scFv-human IgG1 Fc conjugates, respectively. The expressed scFv fusion proteins were purified to apparent homogeneity with yields of approximately 1 and 48 mg, from 1 L of bacterial culture and myeloma cell media, respectively. Surface plasmon resonance and ELISA analyses confirmed that purified scFv proteins showed Man3 specificity. The humanized antibody in scFv-Fc form, derived from clone 5A3, was a disulfide-liked dimer with a molecular mass of 108 kDa. According to a bivalent model, the kinetics parameters of its binding to Man3 were determined to be ka = 4.03 x 104 M-1 s-1, kd = 5.77 x 10-4 s-1, KA = 6.98 x 107 M-1, and KD = 1.43 x 10-8 M. This study thus established the foundation for isolation of carbohydrate-specific scFv genes and eventual production of humanized scFv-Fc type antibodies.

Construction of a recombinant single chain antibody recognizing nonreducing terminal mannose residues applicable to immunohistochemistry.

We recently reported characterization of 25 clones isolated from a phage library displaying human scFvs using a neoglycolipid Man3-DPPE, which was synthesized from mannotriose (Man3) and dipalmitoylphosphatidylethanolamine (DPPE). Of those, 5A3 scFv was successfully expressed and purified as a humanized scFv-Fc form (Sakai et al., Biochemistry 46:253, 2007, Zhang et al. ibid 263). To carry out immunohistochemistry (IHC) in human tissues, a HA tag sequence was introduced to the 5A3 scFv-Fc gene and the resulting construct was transfected to murine myeloma NS0 cells. The 5A3 scFv-Fc protein expressed was affinity-purified. Sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing and reducing conditions and enzyme-linked immunosorbent assay confirmed that 5A3 scFv-Fc protein is dimeric and retained the ability to recognize nonreducing terminal mannose residues. IHC staining of non-neoplastic tissues by this recombinant antibody revealed that no immunoreactivity was detectable in most of 16 tissues examined. Exceptions were found in IHC staining of kidney and pancreas, which demonstrated clear staining of proximal tubules and islet of Langerhans, respectively. These results demonstrated that nonreducing terminal mannose residues are not usually present under normal physiological conditions. This study thus provided a potentially useful tool for examination of the nonreducing terminal mannose residues, which may become exposed under certain pathophysiologycal conditions.

Effects of the surface pressure on the formation of Langmuir-Blodgett monolayer of nanoparticles.

The effects of the surface pressure on the particle arrangement of Langmuir-Blodgett (LB) monolayers of alkanethiol-capped gold nanoparticles were studied. The LB monolayers were prepared from a highly concentrated particle solution, which increases film fabrication efficiency but readily causes small particle voids in the particle array. Overcompressing the LB monolayer to a high surface pressure restructured the particles and eliminated the voids. When the gold particles capped by dodecanethiol were 8.5 nm in diameter, the particle arrangement was vastly improved and a wafer-scale LB monolayer was transferred onto a substrate at the surface pressure of 20 mN/m.