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Lenvatinib (an inhibitor of vascular endothelial growth factor (GF) receptors 1–3, fibroblast GF receptors 1–4, platelet-derived GF receptor α, rearranged during transfection, and stem cell factor receptor) was non-inferior to sorafenib in a phase 3 (REFLECT) trial of advanced hepatocellular carcinoma. This study examined the efficacy and safety of lenvatinib in a real-world setting. This was a retrospective, multicenter, observational study. Inclusion and exclusion criteria were based on the phase 3 trial, and participants were observed for at least 12 weeks. Therapeutic effect was determined using the modified Response Evaluation Criteria In Solid Tumors (m-RECIST) at the 8th week. Patients received oral lenvatinib 12 mg/day (body weight > 60 kg) or 8 mg/day (body weight

Reelin plays an important role in the migration of embryonic neurons, but its continuing presence suggests additional functions in the brain. We now report a novel function where reelin protects P19 embryonal cells from apoptosis during retinoic acid-induced neuronal differentiation. This increased survival is associated with reelin activation of the phosphatidyl-inositol-3-kinase (PI3 K)/Akt pathway. When PI3 K was inhibited with LY294002, reelin failed to protect against this retinoic acid-induced apoptosis. The protective effect of reelin includes activating the Src-family kinases/PI3 K/Akt pathway which then led to selective phosphorylation of Bcl-2/Bcl-XL associated death promoter (BAD) at serine-136, while the phosphorylation-incompetent mutation of BAD (S136A) suppressed this protection. These and additional studies define a novel pathway where reelin binds apoE receptors, significantly activates the PI3 K/Akt pathway causing phosphorylation of BAD which helps to protect cells from apoptosing, thus serving an important role in promoting the survival of maturing neurons in the brain.

In order to perform transmission measurement of metallic sodium in the mm thickness range, we made the sample using a glove box which is quite different technique compared with the previous works. Using a CW source, we have made direct measurement of actual transmittance of a sodium sample with a thickness of 1 ~ 8 mm in the spectral range > 115 nm which is restricted by magnesium fluoride (MgF2) windows, resulting in, for example, ~ 50 % transmittance of a 3 mm thick solid sodium sample including the transmittance of the windows and the reflection losses at each interface at the wavelength of ~ 120 nm, with a very weak temperature dependence up to 150 °C which is beyond the melting point of 97 °C. To confirm the high transmittance, we set up a simple transmission imaging configuration with a 8-mm thick sodium sample, resulting in clear images composed of 100 µm diameter tungsten bars recorded on a Charge Coupled Device detector. The results show that a short pulse intense source such as high order harmonics as well as short pulse EUV plasma source opens a new technique for investigating dynamics of phase transitions as well as hydrodynamics of molten metal.

Electrostatic repulsive force among human red blood cells (RBCs) was observed by macromolecules-induced RBC aggregation and RBC flow behavior in narrow tube, by reducing the sialic acid content of RBCs with neuraminidase. The electrophoretic mobility of the RBCs was proportional to the sialic acid content. (1) When the sialic acid content was reduced, the RBC aggregation observed with a low shear rheoscope was enhanced with fibrinogen (MW=340,000), Ig G (MW=160,000) or Dextran T-70 (MW=70,400), but did not without the macromolecules. In coexistence of normal cells, sialic acid-reduced cells settled significantly faster, possibly due to their preferential aggregation by the macromolecules. (2) Sialic acid-reduced RBCs accumulated more to flow axis in narrow tube (20-50 μm in inner diameter), especially at acidic pH, as evaluated by the thickness of marginal cell-free layer using an image processor. The thickness was further increased with Dextran T-70 due to the RBC aggregation. Conclusively, electrostatic repulsive force among RBCs provided by sialic acid is important for understanding hemorheological behavior of the RBCs and circulatory disturbances in various diseases.

The effects of erythrocyte aggregation on O2release were examined using O2-permeable fluorinated ethylenepropylene copolymer tubes (inner diameter, 25 μm; outer diameter, 100 μm). Measurements were performed using an apparatus built on an inverted microscope that contained a scanning-grating spectrophotometer with a photon count detector connected to two photomultipliers and an image processor through a video camera. The rate of O2release from the cells flowing in the narrow tube was determined based on the visible absorption spectrum and the flow velocity of the cells as well as the tube size. When the tube was exposed to nitrogen-saturated deoxygenated saline containing 10 mM sodium dithionite, the flowing erythrocytes were deoxygenated in proportion to the traveling distance, and the deoxygenation at a given distance increased with decreasing flow velocity and cell concentration (hematocrit). Adding Dextran T-70 to the cell suspension increased erythrocyte aggregation in the tube, which resulted in suppressed cell deoxygenation and increased marginal cell-free-layer thickness. The deoxygenation was inversely proportional to the cell-free-layer thickness. The relation was not essentially altered even when the medium viscosity was adjusted with Dextran T-40 to remain constant. The rate of O2release from erythrocytes in the tube was discussed in relation to the O2diffusion process. We conclude that the diffusion of O2from erythrocytes flowing in narrow tubes is inhibited primarily by erythrocyte aggregation itself and partly by thickening of the cell-free layer.

The present study was designed to evaluate the effects of free iron on normal red blood cells (RBCs) and possible mechanisms responsible for the alteration of the RBC rheological properties. Human RBCs (Ht 5%) were incubated for 1 h at 37°C with 0–2 mM FeSO 4 in the presence of ascorbate. Thiobarbituric acid reactive substances assay was employed to estimate lipid peroxidation and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate to analyze membrane proteins. A cone-plate viscometer, and low-shear and high-shear rheoscopes were used to compare the rheological parameters in Fe 2+ /ascorbate-treated RBCs with those of diamide-treated ones. The incubation of RBCs with free iron caused a dose-dependent increase in lipid peroxidation, enhanced binding of hemoglobin to the cell membrane, diminishing of band 3 content and the concomitant increase in lower molecular weight peptides (approximately 60 and 35 kDa), while the incubation with diamide resulted in spectrin crosslinking. Both diamide and Fe 2+ /ascorbate treatment impaired RBC deformability and aggregation. The suspensions of Fe 2+ -treated RBCs manifested increased viscosity, presumably due to enhanced formation of three-dimensional aggregates. In conclusion, although in both these cases of oxidative stress the ultimate result was the deterioration of membrane structure and functions, the mechanisms of oxidative changes of the RBCs rheological properties were apparently dissimilar.

Thiol-compounds, dithiothreitol (DTT) and N -acetylcysteine (NAC), were examined for their antioxidant effects on Fe 2+ /ascorbate-treated and diamide-treated red blood cells (RBCs). Both of the compounds protected RBCs against diamide-induced spectrin crosslinking; they also prevented loss of deformability and aggregation impairment in diamide-treated cells. Neither DTT nor NAC were effective in suppressing lipid peroxidation induced by Fe 2+ /ascorbate treatment. DTT showed a limited protective effect on iron-mediated band 3 fragmentation and RBC deformability, but it was capable of preventing the decrease of rouleaux formation rate to some extent. NAC exhibited a dose-dependent band 3 protective activity, partially prevented Fe 2+ /ascorbate-induced deformability impairment and the decrease of rouleux formation rate. These findings suggest that thiol compounds are effective in modulating diamide-induced oxidative alterations of RBCs membrane constituents and rheological properties. NAC is more efficacious than DTT in protecting RBCs against iron-mediated oxidative injury, which can be attributed to its potent reductant and radical scavenging abilities.

The effects of supplemental night lighting and nitrate exclusion in the nutrient solution on the growth, nitrate concentration, and nitrate reductase activity (NRA) in the leaf of spinach plants grown under NFT and natural light in the greenhouse were studied. When the plants became of marketable size, they were grown in a nutrient solution without nitrate and exposed to artificial lighting of different light intensities and qualities from 23 : 00 to 6 : 00 1. Two days after treatments, top fresh weight was not affected by nitrate exclusion, whereas 7 days after treatments, plant growth was markedly reduced by nitrate exclusion. However, supplemental night lighting after nitrate exclusion on the subsequent growth of spinach were ineffective. 2. After nitrate exclusion, sap nitrate concentrations of leaf blades and petioles under supplemental night lighting decreased more rapidly than that without the treatment. The decrease of sap nitrate concentrations was unaffected with light quality. However, light intensities affected markedly, and the sap nitrate concentrations of leaf blade at two days after treatment were 1, 059, 799, 612 and 531 ppm under 0 (without lighting), 50, 150 and 250 m mol m-2s-1 (PPFD), respectively. 3. NRA of the plants with supplemental night lighting was higher than that without lighting.

Metallic sodium (Na) was proposed as a transparent material in the vacuum ultra-violet (VUV) spectral range in 1930s and in 1960s. However no clear transmission has ever been demonstrated. In this paper we describe firstly the direct measurement of actual transmittance of a sodium samples in a spectral range longer than 115 nm which corresponds to the shortest transmission wavelength of magnesium fluoride (MgF 2 ) windows, resulting in several tens of % transmittance of a 3 mm-thick solid sodium sample including MgF 2 windows at the wavelength of ~120 nm. We also find very weak temperature dependency of the transmittance up to 150 degrees centigrade where the solid sample is melted at 97 degrees. The measured transmittance pushes us to make a simple imaging experiment illuminated by the VUV light through a 2-mm thick sodium sample, resulting in obtaining a clear image composed of 100 μm diameter tungsten mesh recorded on a two dimensional Charge Coupled Device detector. The result also opens a way to construct an optical imaging device for objects inside or through a solid or a liquid sodium medium. According to the present experiment, we can make a continuous real time transmission imaging for a liquid sodium sample if we use proper optical setup including an intense continuous VUV source or high repetition rated intense coherent source for holographic data acquisition. Such an experiment opens up a way to perform transmission imaging through or inside a sodium medium for characterization of hydrodynamic and material properties.

CPD blood, in glass containers and polyvinyl chloride (PVC) bags, was X-irradiated with 50 Gy (n = 4), and then stored for up to 4 weeks. Rheological, hematological and biochemical changes of the red cells during storage were examined. Changes in blood viscosity and various properties of red cells (deformability, shape, hematological indices and concentrations of 2,3-diphosphoglycerate and nucleotides) during storage at 4 degrees C were not altered by the irradiation. The rheological, biochemical and morphological properties of red cells were as well preserved in PVC bags as in glass containers. No abnormal and/or cross-linked proteins in red-cell membranes were induced by the irradiation. Irradiation of PVC bags did not affect the above properties of red cells through any changes in the bag constituents. In conclusion, X-ray irradiation with 50 Gy to CPD blood in PVC bags does not affect the rheological functions of red cells.

A phospholipid vesicle that encapsulates a concentrated hemoglobin (Hb) solution and pyridoxal 5′-phosphate as an allosteric effector [Hb vesicle (HbV) diameter, 250 nm] has been developed to provide an O2carrying ability to plasma expanders. The O2release from flowing HbVs was examined using an O2-permeable, fluorinated ethylenepropylene copolymer tube (inner diameter, 28 μm) exposed to a deoxygenated environment. Measurement of O2release was performed using an apparatus that consisted of an inverted microscope and a scanning-grating spectrophotometer with a photon-count detector, and the rate of O2release was determined based on the visible absorption spectrum in the Q band of Hb. HbVs and fresh human red blood cells (RBCs) were mixed in various volume ratios at a Hb concentration of 10 g/dl in isotonic saline that contained 5 g/dl albumin, and the suspension was perfused at the centerline flow velocity of 1 mm/s through the narrow tube. The mixtures of acellular Hb solution and RBCs were also tested. Because HbVs were homogeneously dispersed in the albumin solution, increasing the volume of the HbV suspension resulted in a thicker marginal RBC-free layer. Irrespective of the mixing ratio, the rate of O2release from the HbV/RBC mixtures was similar to that of RBCs alone. On the other hand, the addition of 50 vol% of acellular Hb solution to RBCs significantly enhanced the rate of deoxygenation. This outstanding difference in the rate of O2release between the HbV suspension and the acellular Hb solution should mainly be due to the difference in the particle size (250 vs. 7 nm) that affects their diffusion for the facilitated O2transport.

The oxygen release from flowing erythrocytes under accelerational force (0-4 g) was examined using an oxygen-permeable, fluorinated ethylenepropylene copolymer tube (25 microm in inner diameter). The narrow tube was fixed vertically on the rotating disk of a new centrifuge apparatus, and erythrocyte suspension was perfused in the direction of Earth gravity. The accelerational force was applied perpendicularly to the flow direction of cells by centrifugation. The microscopic images of the flowing cells obtained at five different wavelengths were analyzed, and marginal cell-free layer and oxygen saturation of the cells were measured. By lowering oxygen tension around the narrow tube, erythrocytes were deoxygenated in proportion to their traveling distance, and the deoxygenation was enhanced with decreasing flow velocity and hematocrit. With increase of the g-value, the shift of flowing erythrocyte column to the centrifugal side was increased, the column was compressed, and the oxygen release from the cells was suppressed. Qualitatively, similar results were obtained by inducing erythrocyte aggregation with Dextran T-70 (MW = 70,400), without accelerational force. These results conclude that both the accumulation of erythrocytes under accelerational force and the enhancement of erythrocyte aggregation by macromolecules lead to the reduction of oxygen release from the flowing cells.

Objective Aceruloplasminemia is an iron metabolic disorder caused by mutations in the ceruloplasmin gene. It is characterized by progressive neurodegeneration in association with iron accumulation. Excess iron functions as a potent catalyst of biologic oxidation. Previously we showed that an increased iron concentration is associated with the products of lipid peroxidation in the serum, cerebrospinal fluid, and brain tissues. To clarify the free radical-mediated tissue injury caused by intracellular iron accumulation through mitochondrial dysfunction. Patients and Methods We have measure brain oxygen and glucose metabolisms using positron emission tomography (PET) and examined brains at autopsy for iron contents and activities of the mitochondrial respiratory chain in two affected patients who had different truncation mutations of the ceruloplasmin gene. Results PET showed a marked decrease in glucose and oxygen consumption in the entire brain of aceruloplasminemia patients, with a preponderance of metabolic reduction in basal ganglia. Enzyme activities in the mitochondrial respiratory chain of the basal ganglia were reduced to approximately 45% and 42% respectively for complexes I and IV. An inverse relationship was shown between the amounts of iron accumulated and the levels of mitochondrial enzyme activities in all the brain regions examined. Conclusion Iron-mediated free radicals may contribute to the impairment of mitochondrial energy metabolism in aceruloplasminemia.(Internal Medicine 41: 186-190, 2002)

Electrostatic repulsion among erythrocytes in flow was evaluated through measurement of the thickness of the marginal cell-free layer in narrow glass tubes of 20–50 μ m in inner diameter. To reduce the electrostatic repulsive force, due mainly to sialic acid of the membrane glycoproteins, human erythrocytes were treated with neuraminidase. The surface negative charge of the erythrocytes, as determined from the electrophoretic mobility using free-flow electrophoresis, was found to be proportional to the sialic acid content. When erythrocytes with decreased sialic acid content flowed through narrow tubes, the thickness of cell-free layer determined using an image processor increased even in the absence of erythrocyte aggregation in the suspension. The effect was more pronounced at acidic pH. The addition of Dextran T-70 (70,400 Mol. Wt.) further increased the cell-free layer thickness due to erythrocyte aggregation. Thus, reducing the negative charge density on the erythrocyte surface by itself accelerates the axial accumulation of erythrocytes in flow due to the decreased electrostatic repulsive force between the cells, even in the absence of erythrocyte aggregation.

To construct images of oxygen saturation and the distribution of erythrocytes in a network of microvessels.The image of a small group of microvessels under an inverted microscope was incorporated into an image processor through a video camera and was digitized. Based on the information obtained through six visible interference filters of different wavelengths, oxygen saturation and the amount of erythrocytes in microvessels were calculated with a computer. The system was applied to a microvascular bed of isolated rabbit mesentery perfused with a suspension of human erythrocytes.In a steady flow, with lowering of tissue oxygen tension by superfusion with nitrogen-bubbled isotonic saline containing 20 mmol/L sodium dithionite, the decrease of oxygen saturation of erythrocytes from arterioles to venules was imaged. Simultaneously, the distribution of erythrocytes in the microvessels was imaged and the marginal cell-free layer was profiled under high magnification. No significant alteration of the erythrocyte distribution on the deoxygenation of erythrocytes was observed. The exposure of erythrocytes to acidic pH and the decrease of the flow velocity of erythrocytes increased the release of oxygen from the erythrocytes in microvessels.The present method will be useful for the comprehensive analysis of oxygen transfer in the microvascular network, on the basis of both changes of the oxygen saturation and the flow behavior of erythrocytes.

We have developed a high-level production system for the C-terminal domain of secretory leukoprotease inhibitor (SLPI) to investigate its pharmacological activities. A gene for the C-terminal domain of SLPI, (Asn55-Ala 107)SLPI, was constructed from chemically synthesized deoxyoligonucleotides. It was fused to a gene for the N-terminal portion of human growth hormone via a DNA sequence encoding Leu-Val-Pro-Arg, which can be cleaved by thrombin. The fused gene was expressed in Escherichia coli under the control of a trp promoter, and the fusion protein was obtained as an inclusion body. After sulfonation of the cysteine residues, the sulfonated fusion protein was cleaved at the desired site by thrombin. Sulfonated (Asn55-Ala107) SLPI was refolded in Tris buffer containing reduced and oxidized glutathione. The resulting (Asn55-Ala107) SLPI was purified by cation-exchange chromatography and reverse-phase high performance liquid chromatography. The final yield was 50 mg/I culture. (Asn55-Ala107) SLPI was as active against elastase as, but had less trypsin inhibitory activity than, native SLPI. This system is suitable for the large-scale production of the C-terminal domain of SLPI, which is an elastase-specific inhibitor.

1. In order to characterize the physiological functions of the domain structure of secretory leukoprotease inhibitor (SLPI), the biological capacities of half-length SLPIs, (Ser1-Pro54)SLPI and (Asn55-Ala107)SLPI, were investigated and compared with those of full-length SLPI. 2. The activities of these inhibitors against several serine proteases were determined using synthetic chromogenic substrates. The inhibitory capacity of the C-terminal domain, (Asn55-Ala107)SLPI, was as strong as that of full-length SLPI against human neutrophil elastase (NE), cathepsin G and chymotrypsin. It possessed less trypsin inhibitory activity than intact SLPI. For the N-terminal domain of SLPI, (Ser1-Pro54)SLPI, no inhibitory activity could be detected against the serine proteases tested in this study. 3. The inhibitory activity of (Asn55-Ala107)SLPI against the proteolysis of the natural substrates elastin and collagen by NE was comparable with that of full-SLPI (elastin, IC50 = 907 +/- 31 nM for SLPI, 767 +/- 33 nM for (Asn55-Ala107)SLPI; collagen, IC50 = 862 +/- 36 nM for SLPI, 727 +/- 47 nM for (Asn55-Ala107)SLPI). 4. The binding affinities of full- and half-length SLPIs for heparin were measured by affinity column chromatography. Full-length SLPI showed high affinity for heparin while the binding capacities of both half-length SLPIs were lower. (Concentration of NaCl for elution, 0.45 M for SLPI, 0.24 M for (Ser1-Pro54)SLPI, 0.27 M for (Asn55-Ala107)SLPI). 5. The effects of full-SLPI and (Asn55-Ala107)SLPI on blood coagulation were measured using the activated partial thromboplastin time (APTT). Full-length SLPI prolonged clotting time dose dependently(1.25, 2.5 and 5.0 microM), whereas (Asn55-AlalO7)SLPI had no effect even at the highest concentration.6. In conclusion, the C-terminal domain of SLPI is a promising candidate for the treatment of inflammatory diseases in which participation of neutrophil proteases has been suggested.

Effects of erythrocyte aggregation on the flow dynamics of erythrocytes in microvessels were examined quantitatively by perfusing human erythrocytes suspended in isotonic medium containing various concentrations of dextran (70,400 avg mol wt, Dx-70) into a part of the microvascular bed isolated from rabbit mesentery. Thickness of the marginal cell-free layer was measured with an image analyzer, total flow resistance was determined on the basis of the perfusion pressure-volume flow relationship, and homogeneity of erythrocyte flow was evaluated by the power spectrum obtained by the fast Fourier transform of the light intensity change monitored on single microvessels. With increasing dextran concentration, suspension viscosity of erythrocytes at high shear rates increased linearly and thickness of the cell-free layer increased in a sigmoidal fashion. Flow resistance increased relatively little over the range of dextran concentrations in which the cell-free layer increased most rapidly. Furthermore, the flow pattern of erythrocytes in microvessels became inhomogeneous. In conclusion, the present study shows that Dx-70-induced erythrocyte aggregation results in increased flow resistance in the circulatory system, even through the widening of the cell-free layer tends to reduce the resistance and also results in inhomogeneous flow of erythrocytes in microvessels.

Flow dynamics of human erythrocytes was compared in elastic (E) and hardened (H) microvessels with inner diameters of 10-40 microns. The thickness of the marginal cell-free layer and the overall flow resistance were measured with a vascular bed isolated from rabbit mesentery (E vascular bed) as well as with a 4% paraformaldehyde-fixed bed (H vascular bed). 1) In both E and H microvessels, the thickness of the cell-free layer increased with increasing inner diameter of the microvessels and with decreasing hematocrit accompanied by an overall decrease in the flow resistance. The hematocrit-dependent change of the cell-free layer thickness was greater in the E microvessels than in the H microvessels. The flow resistance was always greater in the H vascular beds than in the E vascular beds. 2) With decreasing erythrocyte deformability induced by treatment with 2 mM diazenedicar-boxylic acid bis(N,N-dimethylamide), the thickness of the cell-free layer decreased at a low hematocrit in the E microvessels and at a high hematocrit in the H microvessels, although the flow resistance was increased in both vascular beds. 3) Dextran of 70,400 average molecular weight accelerated the formation of the cell-free layer by inducing erythrocyte aggregation. A drastic increase in the cell-free layer thickness at 2-4 g/dl of dextran in the E microvessels and at 1-2 g/dl of dextran in the H microvessels was accompanied by a significantly lower increase in the flow resistance. This study concludes that the elasticity of microvessels may play an important role for reducing the overall flow resistance of a vascular bed, which is modulated by the marginal cell-free layer, itself a function of the rheological properties of the erythrocytes.