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Low-temperature radical polymerization of achiral N-allyl-N-tert-butylacrylamide (AltBAAm) was conducted in toluene in the presence of chiral tartrates, such as diethyl l -tartrate (L-EtTar) and di-n-butyl l -tartrate. The 1H NMR spectra of the polymers obtained indicated progress of cyclopolymerization at low temperatures such as −80 °C. Optical properties of the poly(AltBAAm)s examined by optical rotation and circular dichroism measurements indicated asymmetric induction in the cyclopolymerization of achiral AltBAAm. For example, addition of L-EtTar at −80 °C provided the polymer with specific rotation of −4.8°. The tert-butyl groups of poly(AltBAAm)s were removed by treatment with CF3SO3H, transforming into poly(N-allylacrylamide)s [poly(AlAAm)s]. Then, stereochemistry in the stereorepeating unit was investigated by comparing 1H NMR signals of the poly(AlAAm)s with those of model compounds, cis- and trans-3-ethyl-4-methyl-2-pyrrolidones. Taking the negative specific rotation values of the poly(AltBAAm)s obtained with L-tartrates into account, it was revealed that (3S,4S)-trans-unit was predominantly formed through the hydrogen-bond-assisted complex formation of AltBAAm with L-tartrates.

The behavior of environment-assisted cracking (EAC) for super-elastic TiNi alloy was investigated as functions of solution pH and electrochemical potential. The specimen was immersed in sulfate solutions at various pHs, and subjected to a potentiostatic slow strain rate tensile test. As a result, the EAC map for the TiNi alloy as functions of the solution pH and the applied potential was successfully produced, and it was revealed that larger EAC susceptibility was obtained in the region of lower potential and lower pH. The tendency was quite typical for hydrogen embrittlement, but not similar to that of TiAl. The EAC susceptibility was classified by cathodic charge density, irrespective to pH nor potential: The charge density larger than 0.025 MC.m-2 induced a maximum EAC susceptibility.

Microtubules are key components of the cytoskeleton in eukaryotic cells. The dynamics between assembled microtubules and free tubulin dimers in the cytoplasm is closely related to the active shape changes of microtubule networks. One of the most fundamental questions is the association of microtubule dynamics with the molecular conformation of tubulin within microtubules. To address this issue, we applied a new technique for the rapid shear-flow alignment of biological filaments, enabling us to acquire the structural periodicity data of microtubules by X-ray fiber diffraction under various physiological conditions. We classified microtubules into three main groups on the basis of distinct axial tubulin periodicities and mean microtubule diameters that varied depending on GTP hydrolysis and the content of paclitaxel, a microtubule stabilizer. Paclitaxel induced rapid changes in tubulin axial repeats in a cooperative manner. This is the first demonstration of dynamic changes of axial tubulin repeats within native microtubules without fixation. We also found extraordinary features of negative thermal expansion of axial tubulin repeats in both paclitaxel-stabilized and GMPCPP-containing microtubules. Our results suggest that even in assembled microtubules, both GTP- and GDP-tubulin dimers can undergo dynamic conversion between at least two different states: short and long configurations.

Choline and choline metabolites are essential for all cellular functions. They have also been reported to be crucial for neural development. In this work, we studied the functional characteristics of the choline uptake system in human neural stem cells (hNSCs). Additionally, we investigated the effect of extracellular choline uptake inhibition on the cellular activities in hNSCs. We found that the mRNAs and proteins of choline transporter-like protein 1 (CTL1) and CTL2 were expressed at high levels. Immunostaining showed that CTL1 and CTL2 were localized in the cell membrane and partly in the mitochondria, respectively. The uptake of extracellular choline was saturable and performed by a single uptake mechanism, which was Na+-independent and pH-dependent. We conclude that CTL1 is responsible for extracellular choline uptake, and CTL2 may uptake choline in the mitochondria and be involved in DNA methylation via choline oxidation. Extracellular choline uptake inhibition caused intracellular choline deficiency in hNSCs, which suppressed cell proliferation, cell viability, and neurite outgrowth. Our findings contribute to the understanding of the role of choline in neural development as well as the pathogenesis of various neurological diseases caused by choline deficiency or choline uptake impairment.

Colloid transport experiments focusing on the initial deposition stage in water-unsaturated sand columns were conducted. To examine the effect of electrostatic interaction in the unsaturated condition, negatively and positively charged silica colloids were used for column transport experiments under different salt concentrations. The results of the column experiments were analyzed based on the colloid filtration theory and the deposition rate constants, and the single collector efficiency was calculated. The deposition rate constants of both negatively and positively charged silica in a water-unsaturated condition are larger than those in a water-saturated condition at an equivalent salt concentration, because the interface between air and water acts as an additional deposition site. The negatively charged silica shows the salt concentration, above which electric double layer (EDL) repulsion can be neglected, and the salt concentration is called critical deposition concentration (CDC). The CDCs were almost the same values in water-saturated as well as unsaturated conditions. The deposition rate constants of the positively charged silica were slightly increased at 0.05 mM due to the EDL attractive forces in the saturated condition. However, we could not see the significant effects of the EDL attractive force in the unsaturated condition in this study. Also, the present results demonstrated that a correlation equation for calculating collector efficiency can be applied to the non-spherical collector particles.

G-protein-coupled receptors (GPCRs) may form heteromeric complexes and cooperatively mediate cellular responses. Although heteromeric GPCR complexes are suggested to occur in many neurons, their contribution to neuronal function remains unclear. We address this question using two GPCRs expressed in cerebellar Purkinje cells: adenosine A1 receptor (A1R), which regulates neurotransmitter release and neuronal excitability in central neurons, and type-1 metabotropic glutamate receptor (mGluR1), which mediates cerebellar long-term depression, a form of synaptic plasticity crucial for cerebellar motor learning. We examined interaction between these GPCRs by immunocytochemical, biochemical, and Förster resonance energy transfer analyses in cultured mouse Purkinje cells and heterologous expression cells. These analyses revealed that the GPCRs closely colocalized and formed heteromeric complexes on the cell surfaces. Furthermore, our electrophysiological analysis showed that CSF levels (40–400 nm) of adenosine or synthetic A1R agonists with comparable potencies blocked mGluR1-mediated long-term depression of the postsynaptic glutamate-responsiveness (glu-LTD) of cultured Purkinje cells. A similar dose of the A1R agonist decreased the ligand affinity of mGluR1 and did not affect depolarization-induced Ca2+influx, which is an essential factor in inducing glu-LTD. The A1R agonist did not affect glu-LTD mimicked by direct activation of protein kinase C. These results suggest that A1R blocked glu-LTD by decreasing the ligand sensitivity of mGluR1, but not the coupling efficacy from mGluR1 to the intracellular signaling cascades. These findings provide a new insight into neuronal GPCR signaling and demonstrate a novel regulatory mechanism of synaptic plasticity.

Histone acetylation, which alters the compact chromatin structure and changes the accessibility of DNA to regulatory proteins, is emerging as a fundamental mechanism for regulating gene expression. Histone deacetylase (HDAC) inhibitors increase histone acetylation and enhance fear extinction. In this study, we examined whether vorinostat, an HDAC inhibitor, facilitates fear extinction, using a contextual fear conditioning (FC) paradigm, in Sprague-Dawley rats. We found that vorinostat facilitated fear extinction. Next, the levels of global acetylated histone H3 and H4 were measured by Western blotting. We also assessed the effect of vorinostat on the hippocampal levels of NMDA receptor mRNA by real-time quantitative PCR (RT-PCR) and protein by Western blotting. 2 h after vorinostat administration, the levels acetylated histones and NR2B mRNA, but not NR1 or NR2A mRNA, were elevated in the hippocampus. The NR2B protein level was elevated 4 h after vorinostat administration. Last, we investigated the levels of acetylated histones and phospho-CREB (p-CREB) binding at the promoter of the NR2B gene using the chromatin immunoprecipitation (ChIP) assay followed by RT-PCR. The ChIP assay revealed increases in the levels of acetylated histones and they were accompanied by enhanced binding of p-CREB to its binding site at the promoter of the NR2B gene 2 h after vorinostat administration. These findings suggest that vorinostat increases the expression of NR2B in the hippocampus by enhancing histone acetylation, and this process may be implicated in fear extinction.

We have studied the transport of colloidal silica in various degrees of a water-saturated Toyoura sand column, because silica particles are widely used as catalyst carriers and abrasive agents, and their toxicity is reported recently. Since water-silica, water-sand, and air-water interfaces have pH-dependent negative charges, the magnitude of surface charge was controlled by changing the solution pH. The results show that, at high pH conditions (pH 7.4), the deposition of colloidal silica to the sand surface is interrupted and the silica concentration at the column outlet immediately reaches the input concentration in saturated conditions. In addition, the relative concentration of silica at the column outlet only slightly decreases to 0.9 with decreasing degrees of water saturation to 38%, because silica particles are trapped in straining regions in the soil pore and air-water interface. On the other hand, at pH 5 conditions (low pH), where sand and colloid have less charge, reduced repulsive forces result in colloidal silica attaching onto the sand in saturated conditions. The deposition amount of silica particles remarkably increases with decreasing degrees of water saturation to 37%, which is explained by more particles being retained in the sand column associated with the air-water interface. In conclusion, at higher pH, the mobility of silica particles is high, and the air-water interface is inactive for the deposition of silica. On the other hand, at low pH, the deposition amount increases with decreasing water saturation, and the particle transport is inhibited.

Microtubules are key components of the cytoskeleton in eukaryotic cells. The dynamics between assembled microtubules and free tubulin dimers in the cytoplasm is closely related to the active shape changes of microtubule networks. One of the most fundamental questions is the association of microtubule dynamics with the molecular conformation of tubulin within microtubules. To address this issue, we applied a new technique for the rapid shear-flow alignment of biological filaments, enabling us to acquire the structural periodicity data of microtubules by X-ray fiber diffraction under various physiological conditions. We classified microtubules into three main groups on the basis of distinct axial tubulin periodicities and mean microtubule diameters that varied depending on GTP hydrolysis and the content of paclitaxel, a microtubule stabilizer. Paclitaxel induced rapid changes in tubulin axial repeats in a cooperative manner. This is the first demonstration of dynamic changes of axial tubulin repeats within native microtubules without fixation. We also found extraordinary features of negative thermal expansion of axial tubulin repeats in both paclitaxel-stabilized and GMPCPP-containing microtubules. Our results suggest that even in assembled microtubules, both GTP- and GDP-tubulin dimers can undergo dynamic conversion between at least two different states: short and long configurations.

Rhodococcus equi strains of intermediate virulence (IMV) for mice possess a 20kDa protein designated Virulence Associated Protein B (VapB) and a virulence plasmid of 79-100kb, and can be recovered from the submaxillary lymph nodes of pigs. The pathogenicity of such R. equi strains for foals is unknown. In this study, two foals, 42 and 43 days of age, were infected intratracheally with 10(6) and 10(9) cells of R. equi IMV strain A5, respectively. The foal infected with 10(9) cells of strain A5 became clinically ill, with the onset of illness (pyrexia and depression) occurring 21 days after inoculation. R. equi was isolated from the feces and tracheal washings of the foal from 14 to 28 days after inoculation. The foal infected with 10(6) cells of A5 showed no clinical signs, and no R. equi was isolated from any of the samples of feces or tracheal washings during the 28 days of observation. Two foals of 45 and 50 days of age were infected with 10(5) or 10(6) of virulent R. equi ATCC 33701 having 15-17kDa surface proteins designated VapA. Both exhibited severe clinical signs (pyrexia, depression and anorexia) at 12 and 13 days after inoculation. Histopathological examination revealed that strain A5 caused focal granulomatous pneumonia in the foals. R. equi IMV strain A5 was isolated from lung lesions of both foals and from the contents of the intestinal tracts of the foal infected with 10(9) bacteria. These results suggest that IMV R. equi having VapB is less virulent than virulent R. equi having VapA in foals. This finding supports our previous results on the pathogenicities of R. equi strains having these virulence-associated antigens assessed by mouse pathogenicity tests.

Pharmacological agents enhancing fear extinction may be promising tools for the treatment of PTSD. Histone acetylation is involved in memory formation, and histone deacetylase (HDAC) inhibitors increase histone acetylation and subsequently enhance fear extinction. In this study, we examined whether vorinostat, an HDAC inhibitor, facilitated fear extinction, using a contextual fear conditioning (FC) paradigm. We found that vorinostat facilitated fear extinction. Next, the levels of global acetylated histone were measured by Western blotting. We also assessed the effect of vorinostat on the hippocampal levels of NMDA receptor mRNA by real-time quantitative PCR (RT-PCR). The levels of acetylated histone and NR2B mRNA, but not NR1 or NR2A mRNA, were elevated in the hippocampus 2 h after administration of vorinostat. We investigated the levels of acetylated histones and phospho-CREB (p-CREB) binding at the promoter of the NR2B gene using the chromatin immunoprecipitation (ChIP) assay followed by RT-PCR. The levels of acetylated histone and the binding of p-CREB to its binding site at the promoter of the NR2B gene were increased. These findings suggest that vorinostat in conjunction with exposure therapy can be a promising new avenue for the treatment of PTSD.

Given that impairment of fear extinction plays a pivotal role in the pathophysiology of posttraumatic stress disorder (PTSD), drugs that facilitate fear extinction may be useful as novel treatments for PTSD. Histone deacetylase (HDAC) inhibitors have recently been shown to enhance fear extinction in animal studies. Using a single prolonged stress (SPS) paradigm, an animal model of PTSD, we examined whether the HDAC inhibitor vorinostat can facilitate fear extinction in rats, and elucidated the mechanism by which vorinostat enhanced fear extinction, focusing on the N-methyl-d-aspartate (NMDA) receptor signals in the hippocampus. Seven days after SPS, rats received contextual fear conditioning, followed by 2-day extinction training. Vorinostat was intraperitoneally injected immediately after second extinction training session. Contextual fear response was assessed 24 h after vorinostat injection. Hippocampal tissues were dissected 2 h after vorinostat injection. The levels of mRNA and protein tested were measured by RT-PCR or western blotting, respectively. Systemic administration of vorinostat with extinction training significantly enhanced fear extinction in SPS rats as compared with the controls. Furthermore, vorinostat enhanced the hippocampal levels of NR2B and calcium/calmodulin kinase II (CaMKII) α and β proteins, accompanied by increases in the levels of acetylated histone H3 and H4. These findings suggest that vorinostat ameliorated the impaired fear extinction in SPS rats, and this effect was associated with an increase in histone acetylation and thereby enhancement of NR2B and CaMKII in the hippocampus. Our results may provide new insight into the molecular and therapeutic mechanisms of PTSD.

This study compared the effects of 2-week administration of a topical steroid (fluticasone propionate [FP] 100 micrograms twice daily) with placebo in 28 patients with perennial nasal allergy who were allergic to house dust and mites in a double-blind randomized study. The number of inflammatory cells and decidual epithelial cells and concentrations of tryptase and eosinophil cationic protein in nasal lavages, and reactivity of the nasal mucosa to histamine and to antigen were investigated. The topical steroid, FP, significantly inhibited all of these assessments. The degree of improvement of nasal reactivity to histamine significantly correlated with the degree of decrease in eosinophil cationic protein levels.

[1] The Clapeyron slope of the perovskite to post-perovskite phase transition has not been well determined experimentally. Crystal structure of CaIrO3 is equivalent to the MgSiO3 post-perovskite phase (space group: Cmcm) at ambient condition. We have found that perovskite phase (Pbnm) of CaIrO3, which is isostructural with MgSiO3 perovskite, is stable at 1–3 GPa and 1400–1550°C. The perovskite to post-perovskite phase transition in CaIrO3 was determined by using piston-cylinder apparatus at 0.9–3.0 GPa and 1350–1550°C. Results show that phase transition pressure is strongly temperature-sensitive with a Clapeyron slope of +17(±3) MPa/°C. These results, obtained for a suitable analogue material, suggest that post-perovskite phase transition in MgSiO3 also has a large positive value of the Clapeyron slope. Such a pronounced exothermic reaction in the Earth's lowermost mantle will promote the upwelling of high-temperature plumes from the core-mantle boundary region.

A molecular biological analysis of cardiac ion channel should be useful for exploring the cause of sudden death, especially when there was no obvious morphological abnormality in the heart. We performed an autopsy of 42-year-old female who suffered from sudden death 10 hours after awakening from general anesthesia. She had a history of prolonged QTc interval. Pathological examination showed mild dilatation of the left ventricle (LV) and histological interstitial fibrosis in the LV and atrioventricular conduction system. Sequence analysis revealed AT deletion at nucleotide position 1735–1736 bp (ΔAT) of the HREG gene. In ΔAT cDNA-transfected HEK293T cells, HERG channel current was not detected under voltage clamp. When co-expressed with the wild-type (WT) HERG, the ΔAT did not exert dominant negative suppression on WT HERG channel current. Confocal microscopy revealed more reduced protein expression on the cell surface for the ΔAT than for the WT. These results suggest that the ΔAT dose not act as a dominant negative and cannot co-assemble to form functional channels. The long QT-interval in the case of heterozygous for this mutation may be explained by the reduced amount of functional HERG channels incorporated in the heart cell membrane.

A radioimmunoassay was developed in which human antibodies to ragweed compete with 12Ι-labeled rabbit anti-ragweed antibodies for the ragweed antigen coupled to cellulose particles. By means of this test, antibodies to ragweed were determined in 96 sera of treated and untreated patients with ragweed allergy and in 32 sera of nonallergic individuals. Within the applied experimental conditions, it was possible to measure antibodies over a range from 0.4 to 200 μg/ml. In patients receiving immunotherapy, the mean value of the antibodies was 7.2 μg/ml, and in the untreated patients 1.8 μg/ml. Antibodies to ragweed were also found in the sera of nonallergic individuals. It is assumed that this test measures ‘total’ serum antibodies to ragweed.

Three allergic disorders manifested on the nose were reported in this session. First of all, an immediate type (Type I) allergy was examplified by the so-called nasal allergy of Dermatophagoides farinae. In addition to the expression of their clinical picture, effectiveness of the hyposensitization therapy was precisely described, including changes of nasal provocation threshold, PK titer, blocking antibody titer, IgE and IgE antibody value during the period of the therapy. Secondarily, it was speculated that Arthus reaction (Type III) induced by the bacterial infection to the paranasal sinuses was one of the main factors producing chronic sinusitis. This theory was mainly proved by immunohistological examination which showed that antibodies to Staphylococcus aureus or complements were distributed in the infiltrated cells, perivascular regions, basement membrane so on. On the third, clinical pictures and differentiation of Wegener's Granulomatosis (WG) and malignant granuloma of the nose (MGN) were presented. It was concluded that the both were clearly different, since, from immunological and histological findings, WG has characteristics of autoimmune disease and MGN could be classified in malignant lymphoma.

Summary The RAST value obtained in the A. fumigalus system, from sera of patients with allergic bronchopulmonary aspergillosis (ABPA) is dependent not only on the reagin titre but also on blocking factors that depress the RAST score. These factors are primarily specific for A. fumigatus and can be removed by an A. fumigatus immuno-adsorbent. However, some blocking activity could be found in normal sera even after absorption with an A, fumigates immunoadsorbent and seems to be non-specific. All of these blocking factors influence the specificity of the RAST, and complicate the comparison of the quantitation of IgE antibodies in different sera. The use of discs rather than cellulose particles and the use of dilute sera result in a RAST assay more proportional to the specific IgE antibodies to A. fumigates.

Immunoglobulin E-rich fractions and IgE-depleted fractions were prepared from individual and pooled sera of patients allergic to ragweed. The IgE-depleted fractions contained antibodies to ragweed associated with the immunoglobulins G, A and M. The IgE-rich fraction contained only IgE antibodies to ragweed, although other immunoglobulins were present in this fraction. The amount of antibodies in these fractions was determined by the radioimmunoassay described in a preceding publication. The inhibitory role of the non-IgE antibodies was examined in an allergosorbent test (RAST) by using various concentrations of the IgE-rich fraction. The RAST was performed using ragweed-coupled cellulose powder as well as paper discs. The IgE antibodies in concentrations from 0.017 to 0.069 μg/ml were inhibited by 30–60% in the cellulose RAST by non-IgE antibodies in concentrations from 6.25 to 12.5 μg/ml. The inhibition effect was substantially less pronounced in the paper disc RAST. This would indicate that for diagnostic purposes, especially for examination of sera with high levels of IgG antibodies, it is more reliable to use the disc RAST instead of the cellulose powder RAST.

Since 1982, we had used the Jackson-Ono's direct bronchoscope with Tanaka's adapter for HFJV. This device was attached to the proximal end of the bronchoscope, and had a short channel for HFJV. However, this method resulted in high PaCO2 (mean 63.9±20.5mmHg) during long surgical procedure. Therefore, controlled respiration was intermittently required to increase the ventilatory flow per minute.Recently, a bronchoscope with a long built-in channel has been devised at our department, in which spouting orifice of the oxygen is located inside the scope approxymately 6cm proximal from the tip. Since 1986, this device has been applied to 9 patients with bronchial foreign bodies and relatively lower PaCO2 (mean 29.3±11.3mmHg) has been observed during the surgical procedure.Several basic experiments were performed to clarify the mechanism of better clinical results in blood gas analysis using a model of lung and bronchus complex. Following results were obtained: (1) the pressure at the distal end of the newly devised bronchoscope was significantly higher; (2) the ventilatory flow rate per minute was significantly higher than that of conventional bronchoscope with Tanaka's adapter; (3) there was no significant difference in airway pressure between these two instruments. From these results we concluded that the reason why low PaCO2 could be maintained with newly developed bronchoscope during long manipulation was due to high ventilatory flow rate in spite of the similar airway pressure inside the bronchus.

The presence of IgE, IgG and ragweed antigen in or on eosinophils in the nasal secretions of ragweed-sensitive patients was investigated by radioautography using specifically purified 125I-labelled antibodies to the above substances. The results indicate a significant (p

Until recently, there has not been a sufficient number of cases of salivary gland cancer to permit the development of a clinically useful TNM classification system. A comprehensive retrospective study of 354 patients with cancer of the parotid gland from 46 institutions in Japan was carried out at the request of the Japanese Committee on TNM Classification (JJC), and we propose JJC T-categories for parotid gland cancer. The characteristic of this proposal involves T-categories determined according to tumor size. Fixed tumors or tumors with facial nerve involvement were classified as T4 regardless of tumor size. In 1983, the AJC proposal for a TNM classification system of salivary gland cancers was tentatively approved at a AJC/UICC joint meeting. In comparison with the AJC classification, our proposal seems to be better in terms of simplicity and the balanced distribution in T-categories of parotid gland cancers.