Effects of Pain Relief Through Minimal Exercise Intervention in a Rat Model of Neuropathic Pain.

We aimed to minimize the frequency of exercise intervention and test the efficacy of pain relief. We also investigated the mechanism of neuropathic pain to determine the best frequency of pain relief for neuropathic pain. The chronic constriction injury (CCI) rat model was randomly divided into three groups: exercise (Ex), No-Ex, and normal. The treadmill exercise intervention was administered, and the 50% withdrawal threshold was assessed using the Von Frey Test. Ionized calcium-binding adaptor molecule 1 (IBA1), glial fibrillary acidic protein (GFAP), brain-derived neurotrophic factor (BDNF), C-C chemokine receptor type 2 (CCR2), and tumor necrosis factor receptor-associated factor 6 (TRAF6) activation was determined through immunohistochemistry. In the brain, we examined the increased expression of ß-endorphin/met-enkephalin in the gray matter of the midbrain aqueduct. Co-expression of CCR2, IBA1, and Neu-N was observed in the spinal cord dorsal horn by immunofluorescence staining. The 50% pain response threshold was significantly lower in the Ex group than in the No-Ex group at five weeks post-CCI, indicating a high analgesic effect. In the dorsal horn of the spinal cord, IBA1 and GFAP were significantly decreased in the Ex group than in the No-Ex group at five weeks post-CCI. However, no significant difference in activation of BDNF, CCR2, and TRAF6 was observed. In the midbrain, the Ex group showed a significant increase compared to the No-Ex group. In summary, our results suggest that in minimal-exercise intervention, neuropathic pain relief is achieved by activation of the descending pain inhibitory system in the midbrain.

1,5-anhydro-D-fructose induces anti-aging effects on aging-associated brain diseases by increasing 5'-adenosine monophosphate-activated protein kinase activity via the peroxisome proliferator-activated receptor-γ co-activator-1α/brain-derived neurotrophic factor pathway.

5'-Adenosine monophosphate-activated protein kinase (AMPK) is a metabolic sensor that serves as a cellular housekeeper; it also controls energy homeostasis and stress resistance. Thus, correct regulation of this factor can enhance health and survival. AMPK signaling may have a critical role in aging-associated brain diseases. Some in vitro studies have shown that 1,5-anhydro-D-fructose (1,5-AF) induces AMPK activation. In the present study, we experimentally evaluated the effects of 1,5-AF on aging-associated brain diseases in vivo using an animal model of acute ischemic stroke (AIS), stroke-prone spontaneously hypertensive rats (SHRSPs), and the spontaneous senescence-accelerated mouse-prone 8 (SAMP8) model. In the AIS model, intraperitoneal injection of 1,5-AF reduced cerebral infarct volume, neurological deficits, and mortality. In SHRSPs, oral administration of 1,5-AF reduced blood pressure and prolonged survival. In the SAMP8 model, oral administration of 1,5-AF alleviated aging-related decline in motor cognitive function. Although aging reduced the expression levels of peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) and brain-derived neurotrophic factor (BDNF), we found that 1,5-AF activated AMPK, which led to upregulation of the PGC-1α/BDNF pathway. Our results suggest that 1,5-AF can induce endogenous neurovascular protection, potentially preventing aging-associated brain diseases. Clinical studies are needed to determine whether 1,5-AF can prevent aging-associated brain diseases.

Effects of low-intensity exercise on spontaneously developed knee osteoarthritis in male senescence-accelerated mouse prone 8.

BACKGROUND: Osteoarthritis (OA) is a degenerative joint disease associated with aging, which often leads to joint stiffness and disability. Exercise is one of the most important non-pharmacological treatments and is prescribed as an indispensable treatment for OA. However, whether physical exercise is beneficial for preventing the progression of OA symptoms with age is poorly understood. We investigated the effects of exercise on spontaneously developed knee OA using male senescence-accelerated mouse prone 8 (SAMP8). METHODS: To examine age-related changes in the knee joints of SAMP8, knee articular cartilage changes, synovitis, knee joint flexion and extension angles, swelling, walking ability, and quadriceps muscle atrophy were analyzed at 3, 5, 7, and 9 months. SAMP8 were required to run at a speed of 10 m/min for 15 min/day from 7 to 9 months of age. The knee joint pathologies and symptoms of exercising and non-exercising mice were compared by histological, immunohistochemical, and morphometrical analyses. RESULTS: The mice presented with various histological changes, including cartilage destruction, osteocyte formation, synovitis, declined joint angles, and swelling. Notably, medial and posterior cartilage destruction was more severe than that of the lateral and anterior cartilage. Knee joint angles were significantly correlated with the histological scores (modified Mankin and OARSI, osteophyte formation and synovial lining cell layer). Exercise did not attenuate cartilage degeneration in the medial and posterior tibial plateau, although the articular cartilage of the anterior and lateral tibial plateau and its histological scores was remained and significantly improved, respectively, by exercise. Exercise suppressed the age-related decline of collagen type II-positive areas in the remaining articular cartilage and improved the OA symptoms. Exercise reduced the expression of monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor (TNF)-α positive macrophages in the synovium. CONCLUSION: This study revealed that SAMP8 developed spontaneous knee OA with age, which resembled the disease symptoms in humans. Low-intensity exercise temporarily alleviated degeneration of the remaining cartilage, synovitis, and age-related decreases in knee flexion angle, stride length, and muscle atrophy in SAMP8. However, exercise during OA progression with age may cause mechanical stress that could be both beneficial and detrimental to joint health.

Effects of different remote ischemia perconditioning methods on cerebral infarct volume and neurological impairment in rats.

Remote ischemic perconditioning (RIPerC) is a novel neuroprotective method against cerebral infarction that has shown efficacy in animal studies but has not been consistently neuroprotective in clinical trials. We focused on the temporal regulation of ischemia-reperfusion by RIPerC to establish an optimal method for RIPerC. Rats were assigned to four groups: 10 min ischemia, 5 min reperfusion; 10 min ischemia, 10 min reperfusion; 5 min ischemia, 10 min reperfusion; and no RIPerC. RIPerC interventions were performed during ischemic stroke, which was induced by a 60-min left middle cerebral artery occlusion. Infarct volume, sensorimotor function, neurological deficits, and cellular expressions of brain-derived neurotrophic factor (BDNF), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and caspase 3 were evaluated 48 h after the induction of ischemia. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) was also performed. RIPerC of 10 min ischemia/10 min reperfusion, and 5 min ischemia/10 min reperfusion decreased infarct volume, improved sensorimotor function, decreased Bax, caspase 3, and TUNEL-positive cells, and increased BDNF and Bcl-2 expressions. Our findings suggest RIPerC with a reperfusion time of approximately 10 min exerts its neuroprotective effects via an anti-apoptotic mechanism. This study provides important preliminary data to establish more effective RIPerC interventions.

Stimulation of functional recovery via neurorepair mechanisms by the traditional Japanese Kampo medicine, Ninjin'yoeito, and physical exercise in a rat ischemic stroke model.

ETHNOPHARMACOLOGICAL RELEVANCE: Ninjin'yoeito (NYT), a traditional Japanese Kampo medicine consisting of 12 herbs, has been reported to improve cognitive dysfunction, depression, and neurological recovery in patients with neurovascular diseases such as Alzheimer's disease and stroke. Several studies have reported that the NYT components exert neurotrophic, neurogenic, and neuroprotective effects. In addition, exercise enhances neuroprotection and functional recovery after stroke. Rehabilitative exercises and pharmacological agents induce neurophysiological plasticity, leading to functional recovery in stroke patients. These reports indicate that NYT treatment and exercise may promote functional recovery following stroke through their beneficial effects. However, no study has determined the effects of NYT and the possible mechanisms of neurorepair and functional recovery after stroke. AIM OF THE STUDY: This study aimed to investigate the combined effects of NYT and exercise on neuroprotection and functional recovery and the underlying mechanisms in a rat ischemic stroke model. MATERIALS AND METHODS: Stroke was induced with 60-min middle cerebral artery occlusion (MCAO) followed by reperfusion in adult male Sprague-Dawley rats. After stroke, the rats were assigned to four groups: ischemia reperfusion (IR), NYT, exercise (Ex), and NYT + Ex. NYT-treated rats were fed a diet containing 1% NYT one day after stroke. Exercise was performed using a motorized treadmill for 5 days a week (8-15 m/min, 20 min/day), starting 3 days after stroke. The NYT treatment and exercise were continued for 4 weeks after the stroke. Infarct volume, neurological deficits, sensorimotor functions, expression of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase A (TrkA) and B (TrkB), caspase-3 activity, and the p-Akt/Akt ratio were examined by immunohistochemistry and western blotting. RESULTS: Compared to the IR group, all treated groups indicated reduced infarct volumes. The NYT + Ex group showed significantly improved waking time and beam walking score compared with the IR group. The expression of NGF/TrkA/p-TrkA and BDNF/TrkB was significantly increased in the NYT + Ex group compared with those in the IR group, whereas the number of caspase-3 positive cells around the lesion was significantly lower in the NYT + Ex group than in the IR group. In addition, the ratio of p-Akt/Akt was significantly higher in the NYT + Ex group than in the IR group. CONCLUSIONS: This study suggests that NYT in combination with exercise provides neuroprotective effects and improves sensorimotor function by stimulating NGF/TrkA and BDNF/TrkB, and by activating the Akt pathway in ischemic stroke of rats. NYT may be an effective adjunctive agent in post-stroke rehabilitation.

E8002 Reduces Adhesion Formation and Improves Joint Mobility in a Rat Model of Knee Arthrofibrosis.

Knee arthrofibrosis is a common complication of knee surgery, caused by excessive scar tissue, which results in functional disability. However, no curative treatment has been established. E8002 is an anti-adhesion material that contains L-ascorbic acid, an antioxidant. We aimed to evaluate the efficacy of E8002 for the prevention of knee arthrofibrosis in a rat model, comprising injury to the surface of the femur and quadriceps muscle 1 cm proximal to the patella. Sixteen male, 8-week-old Sprague Dawley rats were studied: in the Adhesion group, haemorrhagic injury was induced to the quadriceps and bone, and in the E8002 group, an adhesion-preventing film was implanted between the quadriceps and femur after injury. Six weeks following injury, the restriction of knee flexion owing to fibrotic scarring had not worsened in the E8002 group but had worsened in the Adhesion group. The area of fibrotic scarring was smaller in the E8002 group than in the Adhesion group (p < 0.05). In addition, the numbers of fibroblasts (p < 0.05) and myofibroblasts (p < 0.01) in the fibrotic scar were lower in the E8002 group. Thus, E8002 reduces myofibroblast proliferation and fibrotic scar formation and improves the range of motion of the joint in a model of knee injury.

Preconditioning Exercise in Rats Attenuates Early Brain Injury Resulting from Subarachnoid Hemorrhage by Reducing Oxidative Stress, Inflammation, and Neuronal Apoptosis.

Subarachnoid hemorrhage (SAH) is a catastrophic form of stroke responsible for significant morbidity and mortality. Oxidative stress, inflammation, and neuronal apoptosis are important in the pathogenesis of early brain injury (EBI) following SAH. Preconditioning exercise confers neuroprotective effects, mitigating EBI; however, the basis for such protection is unknown. We investigated the effects of preconditioning exercise on brain damage and sensorimotor function after SAH. Male rats were assigned to either a sham-operated (Sham) group, exercise (Ex) group, or no-exercise (No-Ex) group. After a 3-week exercise program, they underwent SAH by endovascular perforation. Consciousness level, neurological score, and sensorimotor function were studied. The expression of nuclear factor erythroid 2 p45-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), 4-hydroxynonenal (4HNE), nitrotyrosine (NT), ionized calcium-binding adaptor molecule 1 (Iba1), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), interleukin 1ß (IL-1ß), 14-3-3γ, p-ß-catenin Ser37, Bax, and caspase-3 were evaluated by immunohistochemistry or western blotting. The terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling (TUNEL) assay was also performed. After SAH, the Ex group had significantly reduced neurological deficits, sensorimotor dysfunction, and consciousness disorder compared with the No-Ex group. Nrf2, HO-1, and 14-3-3γ were significantly higher in the Ex group, while 4HNE, NT, Iba1, TNF-α, IL-6, IL-1ß, Bax, caspase-3, and TUNEL-positive cells were significantly lower. Our findings suggest that preconditioning exercise ameliorates EBI after SAH. The expression of 4HNE and NT was reduced by Nrf2/HO-1 pathway activation; additionally, both oxidative stress and inflammation were reduced. Furthermore, preconditioning exercise reduced apoptosis, likely via the 14-3-3γ/p-ß-catenin Ser37/Bax/caspase-3 pathway.

E8002 Inhibits Peripheral Nerve Adhesion by Enhancing Fibrinolysis of l-Ascorbic Acid in a Rat Sciatic Nerve Model.

Perineural adhesions leading to neuropathy are one of the most undesirable consequences of peripheral nerve surgery. However, there are currently no widely used compounds with anti-adhesive effects in the field of peripheral nerve surgery. E8002 is a novel, anti-adhesive, multi-layer membrane that contains L-ascorbic acid (AA). Here, we investigated the effect and mechanism of E8002 in a rat sciatic nerve adhesion model. A total of 21 rats were used. Six weeks after surgery, macroscopic adhesion scores were significantly lower in the E8002 group (adhesion procedure followed by nerve wrapping with E8002) compared to the E8002 AA(-) group (adhesion procedure followed by nerve wrapping with the E8002 membrane excluding AA) and adhesion group (adhesion procedure but no treatment). Correspondingly, a microscopic examination revealed prominent scar tissue in the E8002 AA(-) and adhesion groups. Furthermore, an in vitro study using human blood samples showed that AA enhanced tissue-type, plasminogen activator-mediated fibrinolysis. Altogether, these results suggest that E8002 may exert an anti-adhesive action via AA and the regulation of fibrinolysis.

Thermogravimetric Evolved Gas Analysis and Microscopic Elemental Mapping of the Solid Electrolyte Interphase on Silicon Incorporated in Free-Standing Porous Carbon Electrodes.

Free-standing electrodes, which are free from additives (binders and conductive agents) and even current collectors, are useful in terms of both application research and fundamental study. Here, we demonstrate the preparation of binder-free monolithic carbon electrodes embracing Si nanoparticles in their well-defined porous scaffolds via the one-pot sol-gel reaction followed by carbonization. The free-standing electrodes with a thickness of 150 µm work out as a high-areal-density anode for Li-ion batteries, delivering up to ca. 7 mA h cm-2. As the Si content increases, the capacity decay on cycling becomes pronounced, which is likely to associate with the fracturing and pulverization of Si nanoparticles even with the size smaller than 100 nm after long-term cycles. The thermogravimetry-mass spectrometry profile of the cycled electrode corroborates the successive electrolyte decomposition to grow solid electrolyte interphase (SEI) mainly composed of lithium alkylcarbonates, polymeric species, and LiF, rendering the electrode mass nearly double of its original state after 200 cycles. The elemental mapping analysis reveals that LiF is generated inhomogeneously in the monolithic electrodes unlike the other SEI components, resulting in the concentration gradient depending on the distance from a Li counter electrode.

Neuroprotective effects of different frequency preconditioning exercise on neuronal apoptosis after focal brain ischemia in rats.

OBJECTIVE: Preconditioning exercise can exert neuroprotective effects after stroke; however, the effects of exercise intensity, frequency, duration are unknown. We investigated the neuroprotective effect of different frequency preconditioning exercise on neuronal apoptosis after cerebral ischemia in rats. METHODS: Rats were divided into the following five groups: 5 times a week of exercise (5/w-Ex) group, 3 times a week of exercise (3/w-Ex) group, once a week of exercise (1/w-Ex) group, no exercise (No-Ex) group, and intact control (control) group. Rats were made to run on a treadmill for 30 min per day at a speed of 25 m/min for 3 weeks. After the running program, the rats were subjected to 60-min left middle cerebral artery occlusion. Two days after ischemia, the cerebral infarct volume, neurological and motor function, Bcl-2-associated X protein (Bax)/B-cell lymphoma 2 (Bcl-2) ratio, expression of caspase-3, and TUNEL positive cells were examined in the cerebral cortex surrounding the ischemic zone. RESULTS: The 3/w-Ex and 5/w-Ex groups showed significantly reduced infarct volumes compared with the No-Ex group, but the 1/w-Ex group did not. In addition, the 3/w-Ex and 5/w-Ex groups had improved neurological scores and sensorimotor function compared with the No-Ex group. The Bax/Bcl-2 ratio, expression of caspase-3, and TUNEL-positive cells significantly decreased in the penumbra area in the 3/w-Ex or 5/w-Ex groups compared with the No-Ex group. DISCUSSION: Our findings suggested that three times or more per week of high-intensity preconditioning exercise exert neuroprotective effects through the downregulation of the Bax/Bcl-2 ratio and caspase-3 activation after stroke. ABBREVIATIONS: TUNEL: terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick and labeling; MCAO:middle cerebral artery occlusion; BAX:Bcl-2-associated X protein; Bcl-2: B-cell lymphoma 2; TTC: 2,3,5-triphenyltetrazorlium chloride.

Application of a Novel Anti-Adhesive Membrane, E8002, in a Rat Laminectomy Model.

Neuropathic pain after spinal surgery, so-called failed back surgery syndrome, is a frequently observed common complication. One cause of the pain is scar tissue formation, observed as post-surgical epidural adhesions. These adhesions may compress surrounding spinal nerves, resulting in pain, even after successful spinal surgery. E8002 is an anti-adhesive membrane. In Japan, a clinical trial of E8002 is currently ongoing in patients undergoing abdominal surgery. However, animal experiments have not been performed for E8002 in spinal surgery. We assessed the anti-adhesive effect of E8002 in a rat laminectomy model. The dura matter was covered with an E8002 membrane or left uncovered as a control. Neurological evaluations and histopathological findings were compared at six weeks postoperatively. Histopathological analyses were performed by hematoxylin⁻eosin and aldehyde fuchsin-Masson Goldner staining. Three assessment areas were selected at the middle and margins of the laminectomy sites, and the numbers of fibroblasts and inflammatory cells were counted. Blinded histopathological evaluation revealed that adhesions and scar formation were reduced in the E8002 group compared with the control group. The E8002 group had significantly lower numbers of fibroblasts and inflammatory cells than the control group. The present results indicate that E8002 can prevent epidural scar adhesions after laminectomy.

Transparent Ethylene-Bridged Polymethylsiloxane Aerogels and Xerogels with Improved Bending Flexibility.

Transparent, monolithic aerogels with nanosized colloidal skeletons have been obtained from a single precursor of 1,2-bis(methyldiethoxysilyl)ethane (BMDEE) by adopting a liquid surfactant and a two-step process involving strong-acid, followed by strong-base, sol-gel reactions. This precursor BMDEE forms the ethylene-bridged polymethylsiloxane (EBPMS, O2/2(CH3)Si-CH2CH2-Si(CH3)O2/2) network, in which each silicon has one methyl, two bridging oxygens, and one bridging ethylene, exhibiting an analogous structure to that of the previously reported polymethylsilsesquioxane (PMSQ, CH3SiO3/2) aerogels having one methyl and three bridging oxygen atoms. Obtained aerogels consist of fine colloidal skeletons and show high visible-light transparency and a flexible deformation behavior against compression without collapse. Similar to the PMSQ aerogels, a careful tuning of synthetic conditions can produce low-density (0.19 g cm-3) and highly transparent (76% at 550 nm, corresponding to 10 mm thick samples) xerogels via ambient pressure drying by solvent evaporation due to their high strength and resilience against compression. Moreover, EBPMS aerogels exhibit higher bending strength and bending strain at break against the three-point bending mode compared to PMSQ aerogels. This improved bendability is presumably derived from the introduced ethylene-bridging parts, suggesting the potential for realizing transparent and bendable aerogels in such polysiloxane materials with organic linking units.

Facile preparation of silver nanoparticles homogeneously immobilized in hierarchically monolithic silica using ethylene glycol as reductant.

A facile and "green" method was proposed to introduce Ag nanoparticles (Ag NPs) into the hierarchically monolithic silica uniformly in the presence of (3-aminopropyl)-triethoxysilane (APTES) and ethylene glycol. APTES is used to modify the monolith by incorporating amino groups onto the surface of meso-macroporous skeletons, while ethylene glycol is employed as the productive reductant. Ag NPs are homogeneously immobilized in hierarchically monolithic silica after reduction and drying at 40 °C for different duration times, and the embedded amount of Ag NPs can reach 15.44 wt% when treated once. The embedment of Ag NPs increases with the repeat treatment and the APTES amount, without uncontrollable crystalline growth. The surface areas of Ag NPs embedded in silica monoliths after heat treatment at 300 and 400 °C are higher than those before heat treatment. The modification via APTES and the embedment of Ag NPs does not spoil the morphology of monolithic silica, while changing the pore structures of the monolith. A tentative formation process and a reduction mechanism are proposed for the modification, reduction and embedment. Ag NPs embedded in monolithic silica is promising for wide applications such as catalysis and separation.

Fabrication of large-sized silica monolith exceeding 1000 mL with high structural homogeneity.

Reproducible fabrication of the hierarchically porous monolithic silica in a large volume exceeding 1000 mL has been established. By the hydrothermal enlargement of the fully accessible small pores to exceed 50 nm in diameter, the capillary force emerged on solvent evaporation was dramatically reduced, which allowed the preparation of crack-free monoliths with evaporative solvent removal under an ambient pressure. The local temperature inhomogeneity within a reaction vessel in a large volume was precisely controlled to cancel the heat evolved by the hydrolysis reaction of tetramethoxysilane and that consumed to melt ice cubes dispersed in the solution, resulting in large monolithic silica pieces with improved structural homogeneity. Homogeneity of the pore structure was confirmed, both on macro- and mesoscales, using SEM, mercury intrusion, and nitrogen adsorption/desorption measurements. Furthermore, the deviations in chromatographic performance were examined by evaluating multiple smaller monolithic columns prepared from the monolithic silica pieces cut from different parts of a large monolith. All the daughter columns thus prepared exhibited comparable performances to each other to prove the overall homogeneity of the mother monolith. Preliminary results on high-speed separation of peptides and proteins by the octadecylsilylated silica monolith of the above production have also been demonstrated.

Simple 2D-HPLC using a monolithic silica column for peptide separation.

Separation of peptides by fast and simple two-dimensional (2D)-HPLC was studied using a monolithic silica column as a second-dimension (2nd-D) column. Every fraction from the first column, 5 cm long (2.1 mm ID) packed with polymer-based cation exchange beads, was subjected to separation in the 2nd-D using an octadecylsilylated (C18) monolithic sillica column (4.6 mm ID, 2.5 cm). A capillary-type monolithic silica C18column (0.1 mm ID, 10 cm) was also employed as a 2nd-D column with split flow/injection. Effluentof the first dimension (1st-D) was directly loaded into an injector loop of 2nd-D HPLC. UV and MS detection were successfully carried out at high linear velocity of mobile phase at 2nd-D using flow splitting for the 4.6 mm ID 2nd-D column, or with directconnection of the capillary column to the MS interface. Two-minute fractionation inthe 1st-D, 118-second loading, and 2-second injection by the 2nd-D injector, allowed one minute for gradient separation in the 2nd-D, resulting in a maximum peak capacity of about 700 within 40 min. The use of a capillary column in solvent consumption and better MS detectability compared to a larger-sized column. This kind of fast and simple 2D-HPLC utilizing monolithic silica columns will be useful for the separation of complex mixtures in a short time.

Microanalysis for MDR1 ATPase by high-performance liquid chromatography with a titanium dioxide column.

MDR1 is clinically important because it is involved in multidrug resistance of cancer cells and affects the pharmacokinetics of various drugs. Because MDR1 harnesses adenosine 5'-triphosphate (ATP) hydrolysis for transporting drugs, examining the effect on ATPase activity is imperative for understanding the interactions between drugs and MDR1. However, conventional assay systems for ATPase activity are not sensitive enough for screening drugs using purified MDR1. Here we report a novel method to measure ATPase activity of MDR1 using high-performance liquid chromatography equipped with a titanium dioxide column. The amount of adenosine 5'-diphosphate (ADP) produced by the ATPase reaction was determined within 2 min with a titanium dioxide column (4.6 mm ID x 100 mm). The relationship between ADP amount and chromatogram peak area was linear from 5 pmol to 10 nmol. This method made it possible to reduce the amount of purified MDR1 required for a reaction to 0.5 ng, about 1/20th of the conventional colorimetric inorganic phosphate detection assay. This method is sensitive enough to detect any subtle changes in ATPase activity of MDR1 induced by drugs and can be applied to measure ATPase activity of any protein.

Bonelike apatite formation on ethylene-vinyl alcohol copolymer modified with silane coupling agent and calcium silicate solutions.

An ethylene-vinyl alcohol copolymer (EVOH) was treated with a silane coupling agent and calcium silicate solutions, and then soaked in a simulated body fluid (SBF) with ion concentrations approximately equal to those of human blood plasma. A smooth and uniform bonelike apatite layer was successfully formed on both the EVOH plate and the EVOH-knitted fibers in SBF within 2 days. Part of the structure of the resulting apatite-EVOH fiber composite was similar to that of natural bone. If this kind of composite can be fabricated into a three-dimensional structure similar to natural bone, the resultant composite is expected to exhibit both mechanical properties analogous to those of natural bone and bone-bonding ability. Hence, it has great potential as a bone substitute.