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Interaction analysis between small molecules and RNA as well as structure determination of RNA–small molecule complexes will be the clues to search for compounds that bind to specific mRNA or non-coding RNA in drug discovery. In this study, the RNA-binding ability of a fluoroquinolone derivative, KG022, was examined against single-residue bulge-containing hairpin RNAs as RNA models. Nuclear magnetic resonance analysis indicated that KG022 interacts with the RNAs in the vicinity of the bulge residue, with preferring C and G as the bulge residues. The solution structures of the RNA–KG022 complexes showed that the KG022 binds to the RNAs at the bulge-out regions. Each substituent in KG022 interacts with specific position of RNAs around the bulge-out region probably contributing the specificity of the binding. This work provides a novel member for the RNA-targeted small molecules.

In an attempt to evaluate central and peripheral contributions to muscle fatigue, changes in force development and electromyographic (EMG) signal after sustained isometric plantar flexion (IPF) were examined. Sustained IPF of maximal effort was continued until the contraction force was reduced to values of 50% of maximum voluntary contraction (MVC). Maximal IPFs of 3 s durations were done before (pre- IPF) and after (post-IPF) the sustained IPF. Post IPF was done by immediately finished sustained IPF. The contraction force and the EMG signals of the gastrocnemius in pre- and post-IPFs were measured, and the rate of force development (RFD) and the rate of EMG signal development (RED) were calculated. The evoked torque was electrically stimulated by posterior tibial nerve. The peak force, average force, RFDs in post-IPF were significantly smaller than those in pre-IPF. The root mean square and median power frequency (MdPF) of the EMG were not changed in post-IPF. The electrically evoked torque was increased after sustained IPF. These results suggested that the early phase of post-IPF fatigue was the almost purely muscular origin, while the late phase of IPF fatigue contained the influence of central nervous system, possibly due to the difficulty of large motor neurons recruitment after the sustained IPF.

1/2SLPI is a C-terminal domain of SLPI (secretory leukocyte protease inhibitor) which inhibits various serine proteases broadly. The present study is the first X-ray structural report on how 1/2SLPI with P1 Leu strongly inhibits trypsin and how it can inhibit multiple serine proteases., SLPI (secretory leukocyte protease inhibitor) is a 107-residue protease inhibitor which inhibits various serine proteases, including elastase, cathepsin G, chymotrypsin and trypsin. SLPI is obtained as a multiple inhibitor in lung defense and in chronic airway infection. X-ray crystal structures have so far reported that they are full-length SLPIs with bovine α-chymotrypsin and 1/2SLPI (recombinant C-terminal domain of SLPI; Arg58–Ala107) with HNE (human neutrophil elastase). To understand the role of this multiple inhibitory mechanism, the crystal structure of 1/2SLPI with porcine pancreas trypsin was solved and the binding modes of two other complexes compared. The Leu residue surprisingly interacts with the S1 site of trypsin, as with chymotrypsin and elastase. The inhibitory mechanism of 1/2SLPI using the wide primary binding site contacts (from P2′ to P5) with various serine proteases is discussed. These inhibitory mechanisms have been acquired in the evolution of the protection system for acute inflammatory diseases.

In the coherence analysis of muscles, beta bands of 15-35 Hz were reflecting activation of corticospinal tract (CST). The purpose of this study was to investigate the participation of activation of CST during dual task walking by electromyography (EMG) coherence analysis in healthy adults. Six healthy subjects were performed two types walking in a straight; A) normal walking, B) calculation task during walking. The walking performances were recorded using a video camera. The stride and cadence of steps, walking speed were measured from video movies. EMG signals were measured from proximal and distal of tibial anterior, medial gastrocnemius, and soleus muscles of right leg, and these were analyzed integrated EMG and coherence of flexor and extensor muscles. The walking speed, stride, and cadence of dual task condition were decreased compared with normal walking. Integrated EMG of proximal of tibial anterior was decreased in dual task walking. The coherence of EMG activity during dual task walking was not significantly changed flexor and extensor muscles compared to normal walking. These results suggested that the change of walking performance during dual task condition might not be due to decrease of input from CST to motor neurons. In addition, the measurement method of cognitive function during walking was necessary of other way to investigation.

Alterations in the number and size of motoneurons were studied in the medial gastrocnemius (MG) motor nucleus of diabetic rats (12 or 22 weeks after injection of storeptozotocin) and age-matched controls. Each group contained 6 animals. MG motoneurons were retrogradely labeled by dextran-fluorescein and the number and size of cell bodies were examined. Significantly fewer labeled MG motoneurons were found in the 22-week diabetic rats as compared with age-matched control animals. The mean soma diameter of MG motoneurons was significantly smaller in the 12- and 22-week diabetic animals. Furthermore the soma size for 22-week diabetic animals was smaller than for 12-week diabetic animals. The distribution of average soma diameters in the MG nucleus of control animals was bimodal; cells with larger average diameter were presumed to be alpha-motoneurons and those with smaller diameters were presumed to be gamma. Compared to control animals, the number of smaller MG motoneurons was reduced in 12 week diabetic animals. By 22 weeks, diabetic animals had no small MG motoneurons and the size distribution became unimodal. We conclude that there is a significant decrease in the absolute number and size of MG motoneurons in diabetic rats, with the possibility that the decrease occurred predominantly among the smaller gamma-motoneurons.

The effects of isometric antagonist conditioning contraction (ACC) at various durations and intensities on the contractile force, electromyographic (EMG) amplitude, and their rates of rise of elbow flexor muscles were examined in healthy participants. In particular, we focused on the change in the maximum rate of initial force development of agonists (dFagonist/dt max ), which was evaluated by subtracting antagonist force decaying from apparent initial force development. While the ACC caused no statistically significant effect on the average force during elbow flexion, dFagonist/dt max was significantly increased by the ACC of short durations (1–2 s) and large intensities. Similarly, the ACC did not affect the root mean square EMG amplitude of biceps brachii during elbow flexion, but significantly increased the maximum rate of rise of the absolute EMG amplitude (dE/dt max ). These results suggested that facilitating effects of the ACC could be observed in the initial phase of agonist action in healthy participants, and ACC of shorter durations might be more effective. The increased dE/dt max suggested that increased neural activities might contribute to the antagonist conditioned facilitation of force development.

The 1/2SLPI and HNE complex structure was solved at 1.7 Å resolution and compared with the interaction mechanism of elafin., SLPI (secretory leukocyte protease inhibitor) is a 107-residue non-glycosylated protease inhibitor, which inhibits a wide range of serine proteases, trypsin, chymotrypsin, neutrophil elastase, chymase and cathepsin G. X-ray crystallographic analyses have shown that SLPI comprises two separate domains of similar architecture [Grütter, Fendrich, Huber & Bode (1988 ▶), EMBO J. 7, 345–351] and the C-terminal domain interacts with bovine α-chymotrypsin. In order to understand SLPI’s multiple functions against various serine proteases, the complex HNE (human neutrophil elastase) has been co-crystallized with 1/2SLPI (recombinant C-terminal domain of SLPI; Arg58–Ala107), which has a biological activity similar to full SLPI. The 1/2SLPI and HNE complex structure was solved at 1.7 Å resolution, and compared with the interaction mechanism of elafin, which is a specific inhibitor of elastase. It was found that P1 Leu72i and six hydrogen bonds between the main chains in the primary contact region have sufficient ability to inhibit HNE and PPE (porcine pancreatic elastase), and P5 Tyr68i is important in increasing the selectivity of 1/2SLPI against HNE. The mechanisms of the functions of SLPI are relatively unknown, but the current study could help understand the selectivity of SLPI against HNE and PPE.

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor that primarily inhibits the extrinsic pathway of blood coagulation. It is synthesized by various cells and its expression level increases in inflammatory environments. Mast cells and neutrophils accumulate at sites of inflammation and vascular disease where they release proteinases as well as chemical mediators of these conditions. In this study, the interactions between TFPI and serine proteinases secreted from human mast cells and neutrophils were examined. TFPI inactivated human lung tryptase, and its inhibitory activity was stronger than that of antithrombin. In contrast, mast cell chymase rapidly cleaved TFPI even at an enzyme to substrate molar ratio of 1:500, resulting in markedly decreased TFPI anticoagulant and anti-(factor Xa) activities. N-terminal amino-acid sequencing and MS analyses of the proteolytic fragments revealed that chymase preferentially cleaved TFPI at Tyr159-Gly160, Phe181-Glu182, Leu89-Gln90, and Tyr268-Glu269, in that order, resulting in the separation of the three individual Kunitz domains. Neutrophil-derived proteinase 3 also cleaved TFPI, but the reaction was much slower than the chymase reaction. In contrast, alpha-chymotrypsin, which shows similar substrate specificities to those of chymase, resulted in a markedly lower level of TFPI degradation. These data indicate that TFPI is a novel and highly susceptible substrate of chymase. We propose that chymase-mediated proteolysis of TFPI may induce a thrombosis-prone state at inflammatory sites.

Tumor necrosis factor-alpha (TNF-alpha) is a potent multifunctional cytokine that plays a central role in the pathogenesis of many inflammatory diseases. Interleukin-8 (IL-8) is a principle neutrophil chemoattractant and activator in humans. The alveolar macrophage-derived TNF-alpha initiates lung inflammation through its ability to stimulate IL-8 synthesis in airway epithelial cells. Since recent studies demonstrated that the stimulation of epidermal growth factor receptor (EGFR) could induce IL-8 secretion, the involvement of EGFR in TNF-alpha-induced IL-8 secretion in airway epithelium-like NCI-H292 cells was investigated in this study. TNF-alpha and epidermal growth factor (EGF) stimulated IL-8 secretion in a time- and concentration-dependent manner. Inhibition of the EGFR by either an anti-EGFR neutralizing antibody or by its specific inhibitor AG1478 (1 microM) blocked TNF-alpha-induced IL-8 secretion. In addition, TNF-alpha stimulated tyrosine phosphorylation of the EGFR within 5 min after stimulation. Further, TNF-alpha-induced IL-8 secretion was completely inhibited by the neutralizing antibody against amphiregulin (AR), an EGFR ligand, suggesting that TNF-alpha-induced IL-8 secretion was mediated by the AR-EGFR pathway. Furthermore, TNF-alpha stimulated the release of AR in a concentration-dependent manner. Finally, both AR and IL-8 release-induced by TNF-alpha were eliminated by pretreatment with either GM6001, a broad-spectrum inhibitor for metalloprotease, or TAPI-1, relatively selective inhibitor for TNF-alpha converting enzyme (TACE). These findings indicate that metalloprotease-mediated AR shedding and subsequent activation of EGFR play a critical role in TNF-alpha-induced IL-8 secretion from the human airway epithelium-like NCI-H292 cells, and that TACE is one of the most possible candidates for metalloprotease responsible for TNF-alpha-induced AR shedding.

Human airway trypsin-like protease (HAT), a serine protease found in the sputum of patients with chronic airway diseases, is an agonist of protease-activated receptor-2 (PAR-2). Previous results have shown that HAT enhances the release of amphiregulin (AR); further, it causes MUC5AC gene expression through the AR-epidermal growth factor receptor pathway in the airway epithelial cell line NCI-H292. In this study, the mechanisms by which HAT-induced AR release can occur were investigated. HAT-induced AR gene expression was mediated by extracellular signal-regulated kinase (ERK) pathway, as pretreatment of cells with ERK pathway inhibitor eliminated the effect of HAT on AR mRNA. Both HAT and PAR-2 agonist peptide (PAR-2 AP) induced ERK phosphorylation; further, desensitization of PAR-2 with a brief exposure of cells to PAR-2 AP resulted in inhibition of HAT-induced ERK phosphorylation, suggesting that HAT activates ERK through PAR-2. Moreover, PAR-2 AP induced AR gene expression subsequent to protein production in the cellular fraction through the ERK pathway indicating that PAR-2-mediated activation of ERK is essential for HAT-induced AR production. However, in contrast to HAT, PAR-2 AP could not cause AR release into extracellular space; it appears that activation of PAR-2 is not sufficient for HAT-induced AR release. Finally, HAT-induced AR release was eliminated by blockade of tumour necrosis factor alpha-converting enzyme (TACE) by the TAPI-1 and RNA interference, suggesting that TACE activity is necessary for HAT-induced AR release. These observations show that HAT induces AR production through the PAR-2 mediated ERK pathway, and then causes AR release by a TACE-dependent mechanism.

Although chymases are known to exhibit species differences in regard to angiotensin (Ang) II generation and degradation, their properties have never been compared under the same experimental conditions. We analyzed the processing of Ang I by chymases of a variety of species (human chymase, dog chymase, hamster chymase-1, rat mast cell protease-1 [rMCP-1], mouse mast cell protease-4 [mMCP-4]) at physiological ionic strength and under neutral pH conditions. Human chymase generated Ang II from Ang I without further degradation, whereas the chymases of other species generated Ang II, followed by degradation at the Tyr4-Ile5 site in a time-dependent manner. Kinetic analysis showed that in terms of Ang II generating activity (analyzed by cleavage of the Phe8-His9 bond using the model peptide Ang(5-10), Ile5-His6-Pro7-Phe8-His9-Leu10), the chymases ranked as follows: doghumanhamstermouserat (kcat/Km: 18, 11, 0.69, 0.059, 0.030 microM-1min-1), and that in terms of Ang II degrading activity (i.e., cleavage of the Tyr4-Ile5 bond of Ang II), the order was hamsterratmousedog (kcat/Km: 5.4, 4.8, 0.39, 0.29 microM-lmin-1). These results suggest species differences in the contribution of chymases to local Ang II generation and degradation.

We have succeeded in producing monoclonal antibodies directed against a wide variety of epitopes of human chymase by using two different immunogens: a recombinant human chymase-heparin mixture, and chymase alone. Hybridomas were screened by ELISA, and 7 clones were selected based on antibody titers. Epitopes were localized by Western blotting with a C-terminal-deletion series of chymase-GST fusion proteins, and it was possible to use the antibodies for Western blotting and immunohistochemistry. Dot-blot analysis for species specificity revealed that the MAbs bound canine chymase as well as human chymase, and that two of them also bound rodent chymases. These results indicate that the antibodies can be used for various immunological analyses in further investigations of chymase.

Aim: Brain-derived neurotrophic factor (BDNF) reduces plasma glucose levels in obese db/db diabetic mice and is speculated to produce its effects via the hypothalamus, the regulatory centre of satiety and the autonomic nervous system. The potential effect of BDNF directly on peripheral endocrine organs, however, remains to be clarified. Here we report the effects of BDNF on hormonal secretion from pancreatic islets of Langerhans using their isolated culture. Methods and results: In an immunohistochemical study, mouse pancreatic alpha cells were stained specifically with the anti-TrkB (a specific receptor for BDNF) antibody. After 7 days culture of isolated islets of the normal mouse pancreas, 10 ng/ml BDNF decreased the secretion of glucagon per 6 h significantly less than that of the control (p = 0.016). In contrast, there were no significant changes in insulin secretion or glucagon and insulin contents in the islets cultured under the same conditions. In vivo administration of BDNF (10 mg/kg/day) to normal mice for 7 days significantly decreased their food consumption (p

Equiatomic ternary compounds, TbPdIn and DyPdIn, have been studied by means of DC magnetization, AC susceptibility, magnetic relaxation, specific heat and electrical resistivity measurements. The two compounds are ferromagnets below a transition temperature T C =74 K for TbPdIn and T C =38 K for DyPdIn with metastable magnetic properties, behaving as the irreversibility of the temperature dependence of magnetization below a characteristic temperature T ir and the long-time magnetic relaxation behavior below T C when changing the magnetic field at a constant temperature. DyPdIn shows the second phase transition at T N =23 K, caused by the antiferromagnetic coupling. For both compounds, the ground state is not a collinear ferromagnet, but some canted one with antiferromagnetic component. The observed metastable magnetic properties are discussed in terms of domain-wall pinning model. The complex magnetic structures in DyPdIn and TbPdIn are compared with those of isostructural DyNiAl and HoNiAl.

Interleukin (IL)-8, which is involved in inflammatory responses, is produced by a variety of cell types, monocytes/macrophages and neutrophils, in response to inflammatory stimuli including lipopolysaccha ride, IL-1, and tumor necrosis factor α. Here we report the inhibitory effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) on IL-8 production-dates printpubdate="05/03/02" in human whole blood culture. 1,25(OH)2D3 inhibited only the late phase of the biphasic IL-8 production-dates printpubdate="05/03/02" in lipopolysaccharide-stimulated human whole blood. It also effectively inhibited IL-8 production-dates printpubdate="05/03/02" induced by IL-1β compared with that induced by tumor necrosis factor α. IL-8 mRNA expression in IL-1β-stimulated whole blood was found to require de novo protein synthesis. Although monocytes were found to be mainly responsible for IL-1β-induced IL-8 production-dates printpubdate="05/03/02" in whole blood, 1,25(OH)2D3 inhibited IL-8 production-dates printpubdate="05/03/02...

Background. Diabetic nephropathy is histologically characterized by expansion of the glomerular mesangium because of an increase in extracellular matrix (ECM) proteins (fibronectin and type IV (α2) collagen). Transforming growth factor-β1 (TGF-β1) is considered one of the major cytokines involved in the regulation of ECM synthesis and degradation. TGF-β1 is synthesized as an inactive precursor protein and then converted to its active form by proteolytic enzymes such as plasmin. In the present study we examined the effect of tranexamic acid (TA), a plasmin inhibitor, on ECM accumulation in glomeruli of rats with diabetic nephropathy. Methods. The effects of TA were examined by measuring various markers, including changes in kidney weight, albuminuria, glomerular volume, and the expression of glomerular ECM components (fibronectin, type IV (α2) collagen, and TGF-β1) in four groups of rats. The groups were: controls; control rats treated with TA; rats with streptozotocin-induced diabetes (STZ-rats), and STZ-rats treated with TA. Results. Oral administration of TA (300 mg/kg) twice a day had no effect on kidney weight, albuminuria, or glomerular volume in control rats, whereas it caused a slight increase in fibronectin mRNA and a slight decrease in type IV (α2) collagen and TGF-β1 mRNAs. In STZ-rats, TA significantly decreased all the above markers (glomerular volume, 0.96 ± 0.04 × 106μm3 in STZ vs 0.69 ± 0.04 × 106μm3 in STZ + TA: kidney weight, 3.7 ± 0.6 g in STZ vs 3.4 ± 0.6 g in STZ + TA). TA treatment did not influence glucose levels or body weight in either the control rats or the STZ-rats. Conclusions. In contrast to results of a report showing exacerbation of human diabetic retinopathy in patients treated with TA, our findings showed that TA may have a beneficial effect on diabetic nephropathy in STZ-diabetic rats.

The delay-time spectra of 57 Fe nuclear resonant forward scattering in FeS which belongs to the boundary between the charge-transfer and Mott-Hubbard insulators have been observed under high pressure up to about 6 GPa at BL09XU in SPring-8. The observed beat patterns in delay-time spectra are distinguished two different types which correspond to the two different phases. We have successfully applied the maximum-entropy method to analyze the observed delay-time spectra. The frequencies of quantum beats which directly depend on the energy differences between two hyperfine splitting levels of 57 Fe nuclei were extracted by this analysis and then the hyperfine interaction parameters were determined from there extracted frequencies. Since the evaluated magnetic hyperfine field at 0.1 MPa is smaller than that at 2 GPa, the Neel temperature of FeS with a troilite structure increases with pressure.

The de Haas-van Alphen (dHvA) effect has been investigated on high-quality single crystals of FeP in a double helical magnetic state with a 5 c period in the magnetic fields up to 14 T and at tempe...

We describe an immuno-polymerase chain reaction (immuno-PCR) assay for the detection of human angiotensinogen using identical first and second polyclonal antibodies. The reporter DNA was initially generated by PCR amplification using a biotinylated primer, and was bound with streptavidin to biotinylated second antibody. Human recombinant angiotensinogen sandwiched by antibodies was detected by amplifying the reporter DNA using PCR. To reduce the effect of nonspecific amplification, the optimal concentrations of streptavidin and DNA label were determined to be 0.1 mg/l and 0.5 ng/l, respectively. The detection limit of the immuno-PCR assay was 0.1 ng/l, an approximately 2.5 ×10 5 -fold improvement compared with a conventional enzyme-linked immunosorbent assay. These results indicate that a highly sensitive immuno-PCR for human angiotensinogen can be developed even with identical first and second polyclonal antibodies.

We have demonstrated that 1alpha,25-dihydroxyvitamin D(3)-26, 23-lactone analogs, (23S)- and (23R)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647, TEI-9648, respectively), inhibit HL-60 cell differentiation induced by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], but not differentiation caused by all-trans retinoic acid (D. Miura et al., 1999, J. Biol. Chem. 274, 16392). To assess whether the antagonistic actions of TEI-9647 and TEI-9648 in HL-60 cells are related to 1alpha,25(OH)(2)D(3) breakdown, we investigated their effects on catabolism of 1alpha,25(OH)(2)D(3). In HL-60 cells, the C-24 but not the C-23 side-chain oxidation pathway of 1alpha,25(OH)(2)D(3) has been reported. Here we demonstrate that 1alpha,25(OH)(2)D(3) was metabolized both to 24,25,26,27-tetranor-1alpha,23-(OH)(2)D(3) and 1alpha,25(OH)(2)D(3)-26,23-lactone; thus HL-60 cells constitutively possess both the 24- and the 23-hydroxylases. Metabolism of 1alpha, 25(OH)(2)D(3) was strongly suppressed by 10(-7) M TEI-9647 or 10(-6) M TEI-9648. 1alpha,25(OH)(2)D(3) alone slightly induced 24-hydroxylase gene expression by 8 h with full enhancement by 24-48 h; this induction was inhibited by 10(-6) M TEI-9647 and 10(-6) M TEI-9648 (86.2 and 31.9%, respectively) 24 h after treatment. However, analogs of TEI-9647 and TEI-9648 without the 25-dehydro functionality induced 24-hydroxylase gene expression. These results indicate that TEI-9647 and TEI-9648 clearly mediate their stereoselective antagonistic actions independent of their actions to block the catabolism of 1alpha,25(OH)(2)D(3). Therefore, TEI-9647 and TEI-9648 appear to be the first antagonists specific for the nuclear 1alpha,25(OH)(2)D(3) receptor-mediated genomic actions of 1alpha,25(OH)(2)D(3) in HL-60 cells.

We have measured the specific heat of FeS, Fe0.875X, and Co0.875X (X = S,Se) compounds with a NiAs-like structure over the temperature range from 2 to 30 K. It was found that the ground state of FeS has an insulating character. The opening up of a gap in the density of states is probably caused by electron correlation. Two types of superstructure are known to occur due to ordering of vacancies at iron sites in Fe0.875X; one is a 3c structure and the other a 4c structure. The -values obtained for 3c structure are about 10% larger than those of 4c structures. Accordingly, this result indicates that the electronic structure of Fe0.875X should be sensitive to the ordered structure of the vacancies. Since the -values obtained for non-stoichiometric compounds are larger than the calculated ones, there is electron effective-mass enhancement in non-stoichiometric compounds. Moreover comparison of the observed and calculated electronic contributions to the low-temperature specific heat suggests that the mass enhancement in Pauli-paramagnetic Co0.875X is larger than that in ferrimagnetic Fe0.875X.

In this article, x-ray photoelectron spectroscopy (XPS) spectra of iron chalcogenides, FeS, Fe7S8, and Fe7Se8 are presented. FeS (troilite) and Fe7S8 (pyrrhotite) are among the most common iron compounds on earth. FeS is antiferromagnetic below the Neel temperature of TN=593–598 K. Above Tα=420 K, FeS is a metal with an NiAs-type crystal structure, but becomes an insulator with a narrow band gap of 0.04 eV below Tα. This transition known as the α-transition is accompanied by a small displacement of the Fe and S atoms from the regular NiAs-type structure. Fe7S8 and Fe7Se8 are ferrimagnetic metals with Neel temperatures of TN=578 and 423–460 K, respectively. The crystal structures of Fe7S8 and Fe7Se8 are derived from the NiAs-type one by introducing ordered Fe vacancies. The polycrystalline samples were synthesized by the Bridgman method. Clean surfaces were obtained by scraping in situ with a diamond file. All the XPS spectra of the iron chalcogenides are taken at room temperature.

We have measured the de Haas–van Alphen effect at 0.55 K in the field up to 9.5 T for the single crystal of Pauli paramagnetic NiAs. Six dHvA branches have been observed and they show complicated behavior as a function of the field direction which is applied in the symmetry planes (11\bar20), (10\bar10) and (0001). The corresponding cyclotron masses have been also determined from the temperature dependence of the dHvA amplitude. To discuss the experimental results we have performed the electronic band calculation of NiAs for the non-magnetic state by using the full potential LAPW method. The total energy calculated as a function of the lattice parameter a , keeping the ratio c / a as the observed value, becomes minimum at 3.60 A, which agrees very well with the observed one. The Fermi surface is found to consist of two kinds of hole surface around the ΓA axis and one electron surface with six fold symmetry. For each Fermi surface we have calculated dHvA oscillation frequencies and succeeded to explain the...

The electronic structures of the antiferromagnetic semiconductor FeS and ferrimagnetic metals Fe7S8 and Fe7Se8 have been studied by spin-integrated and spin-resolved photoemission spectroscopy and inverse-photoemission spectroscopy. The overall Fe 3d bandwidth in the photoemission spectra is 25-30 % narrower than the density of states (DOS) predicted by first-principles band-structure calculations and is accompanied by an intense tail on the high-binding-energy side, indicating the correlated nature of electrons in the Fe 3d band. Deviation from the band DOS is more significant in Fe7S8 than in Fe7Se8, and in the minority-spin spectra than in the majority-spin spectra. Cluster-model calculation for FeS has shown satellite structures at high binding energies, but the calculated spectral line shape is not in good agreement with experiment compared to the band DOS. By introducing a self-energy correction to the band DOS, we could explain the narrowing of the overall Fe 3d bandwidth and the high-binding-energy tail shape but not for the unusual broadening of the Fe 3d band within ∼ 1 eV of the Fermi level.

We have measured under pressure Mossbauer spectra of FeS and 3c-type up to 16 GPa and x-ray diffraction patterns of up to 11 GPa at room temperature. It is found for that the compressibilities of the lattice parameters exhibit definite anomalies at around 4.5 GPa and that there is no change in the crystal structure up to 11 GPa. Magnetically ordered Mossbauer spectra are observed below 6.5 GPa for FeS and 4.5 GPa for , whereas the spectra above these pressures are typical of a paramagnetic ordering with a quadrupole splitting. A large reduction in the centre shift is observed at these pressures. It is found that there is a distinct steplike feature of the magnetic hyperfine field at 3.5 GPa for FeS. The electronic states of FeS and are deduced from the volume dependences of the centre shift and the magnetic hyperfine field. Below 3.5 GPa for FeS, the electronic state has an insulating character and the electrons on the iron are well localized and thus contribute to the magnetic moment. In the intermediate-pressure range, from 3.5 to 6.5 GPa for FeS and below 4.5 GPa for , the electronic state is like a semimetallic one. Above 6.5 GPa for FeS and 4.5 GPa for , the electronic bandwidth is large enough to cause the state to become metallic and produces a collapse of the iron magnetic moment.

Magnetic circular dichroism (MCD) has been observed at the Fe L2,3 and M2,3 core-absorption edges in ferrimagnetic Fe7S8 and Fe7Se8 using circularly polarized helical undulator radiation. Some differences were observed between the MCD spectra of both compounds. The features of the MCD spectra are interpreted on the basis of a reported band-structure calculation.

Itinerant ferrimagnetic metals Fe7S8 and Fe7Se8 and antiferromagnetic semiconductor FeS have been studied by spin-polarized and spin-integrated photoemission and inverse-photoemission spectroscopy. Comparison with the density of states given by band-structure calculations indicates that the Fe 3d band is narrowed with spectral weight transfer towards higher binding energies and that the intensity at the Fermi level is suppressed. The band narrowing and the spectral weight transfer is reproduced by including the self-energy correction, suggesting the importance of electron correlation. Spin-polarized photoemission spectra of Fe7S8 and Fe7Se8 show negative spin-polarization near the Fermi level and spin-dependent correlation effect.

In order to elucidate roles of the 2'-O-methylation of pyrimidine nucleotide residues of tRNAs, conformations of 2'-O-methyluridylyl(3'----5')uridine (UmpU), 2'-O-methyluridine 3'-monophosphate (Ump), and 2'-O-methyluridine (Um) in 2H2O solution were analyzed by one- and two-dimensional proton NMR spectroscopy and compared with those of related nucleotides and nucleoside. As for UpU and UmpU, the 2'-O-methylation was found to stabilize the C3'-endo form of the 3'-nucleotidyl unit (Up-/Ump-moiety). This stabilization of the C3'-endo form is primarily due to an intraresidue effect, since the conformation of the 5'-nucleotidyl unit (-pU moiety) was only slightly affected by the 2'-O-methylation of the 3'-nucleotide unit. In fact even for Up and Ump, the 2'-O-methylation significantly stabilizes the C3'-endo form by 0.8 kcal/.mol-1. By contrast, for nucleosides (U and Um), the C3'-endo form is slightly stabilized by 0.1 kcal/.mol-1. Accordingly, the stabilization of the C3'-endo form by the 2'-O-methylation is primarily due to the steric repulsion among the 2-carbonyl group, the 2'-O-methyl group and the 3'-phosphate group in the C2'-endo form. For some tRNA species, 2-thiolation of pyrimidine residues is found in positions where the 2'-O-methylation is found for other tRNA species.(ABSTRACT TRUNCATED AT 250 WORDS)

Vitamin D plays an important role in regulating bone and calcium metabolism. The actions of vitamin D are mediated through the nuclear vitamin D receptor (VDR), and gene disruption of the VDR in mice causes skeletal disorders. However, the precise role of the VDR in each stage of osteoblastogenesis is not well understood. To address this issue, we used a biochemical approach to identify an osteoblast-specific coregulator of the VDR. Using a GST-fused VDR ligand-binding domain as bait, proteins associated with liganded VDR were purified from nuclear extracts of HOS osteoblastic cells and compared with those of HeLa cells. Among the interactants identified by mass fingerprinting, CCAAT displacement protein (CDP) was found as a novel ligand-dependent VDR interactant in HOS cells, together with other previously reported DRIP/TRAP complex components. Further biochemical analysis showed that complex formation between the VDR and CDP was distinct from the previously known DRIP/TRAP complex and the p160 family coactivator complexes. Transient expression of CDP potentiated VDR-mediated transcriptional activation in HOS cells. Furthermore, modulation of CDP expression levels in osteoblastic SaM-1 cells affected vitamin D-dependent osteoblast differentiation before the maturation (mineralization) stage. These findings suggest that CDP is a novel differentiation stage-specific coactivator of the VDR in osteoblasts.

Prebeta1-HDL is a minor HDL subfraction that is an initial acceptor of cellular free cholesterol. Prebeta1-HDL is elevated in hypertriglyceridemia, which is exaggerated with postprandial hyperglycemia. We investigated whether the prebeta1-HDL concentration changes postprandially in type 2 diabetic patients and blood glucose (BG) control reduces this change.We examined 9 healthy controls and 20 diabetic patients with poor BG control. Seven blood samples (30 min before and 2 h after each meal, and at midnight) were obtained daily in the poor (poor-GC: n=20) and improved (imp-GC: n=11) glycemic control phases of diabetic patients after intensive insulin therapy and a low-calorie diet.The prebeta1-HDL concentration did not change postprandially in the controls. However, the fasting prebeta1-HDL concentration in the poor-GC phase was 28.3% higher than in the controls (25.4+/-6.8 vs 19.8+/-6.9 mg/l ApoAI, p0.05) and decreased markedly after breakfast (20.9+/-7.7 mg/l ApoAI, p0.01). In the imp-GC phase, the prebeta1-HDL concentration showed no morning surge, as in the controls.Type 2 diabetic patients in the poor-GC phase have high prebeta1-HDL levels in the morning, followed by a gradual reduction until midnight. BG control diminishes this postprandial change. Glucose metabolism may be involved in modulating reverse cholesterol transport in type 2 diabetic patients.

The de Haas-van Alphen (dHvA) effect was measured for the paramagnetic compound CrP with an orthorhombic MnP-type structure using a high quality single crystal. Ten branches of dHvA frequency below 9200T are obtained in the (010) plane at 0.6K. Four frequencies in a field along the symmetry axes agree qualitatively with the theoretical results calculated with an all-electron relativistic LAPW method. The cyclotron mass ratios in two branches along the [100] axis are 4.6 and 3.1, respectively.

Effects of slight La- or Y-doping on the magnetic properties, especially an unusual magnetic phase diagram, of CeP have been studied mainly through specific heat measurements under magnetic fields up to 10 T on (Ce 0.90 La 0.10 )P and (Ce 0.95 Y 0.05 )P. In spite of essentially similar behavior in the paramagnetic phase, a large difference has been observed in the magnetic phase diagram between (Ce 0.90 La 0.10 )P and (Ce 0.95 Y 0.05 )P. The phases suggesting the existence of Γ 8 layers similar to those of CeP are identified for (Ce 0.95 Y 0.05 )P, but not for (Ce 0.90 La 0.10 )P.

The linear and non-linear ac-susceptibilities of the Mott insulator YVO3 were measured using a Hartshorn mutual inductance bridge. It is found by the temperature dependence of ac-susceptibilities that there are two magnetic phase transitions at 72.1 and 112.0 K. Above 112K, the values of linear and non-linear susceptibilities qualitatively consist with the result of the mean-field theory. It is verified that there is a power-law dependence between linear and non-linear susceptibilities, which is predicted by the mean-field theory.

Background Increased cardiac insulin-like growth factor (IGF)-I production is associated with physiological cardiac hypertrophy in athletes, and IGF-I has been recognized as a cardioprotective agent in experimental animal studies. On the other hand, acromegaly which is characterized by an excess of IGF-I has been linked to impaired cardiac function. Methods and Results Both the relationship between the serum levels of IGF-I and brain natriuretic peptide (BNP), which is released from the cardiac ventricles in response to ventricular stress, and that between IGF-I and the concentrations of the plasma amino-terminal propeptide of procollagen type III (P-III-P), which is associated with myocardial fibrosis, were evaluated in 19 patients after surgical treatment for acromegaly. Echocardiography revealed that left ventricular systolic function and dimensions were within normal range in all patients. Significant inverse correlations were found between IGF-I and the BNP (r=-0.5, p=0.02) and P-III-P levels (r=-0.62, p=0.005). Conclusion We observed an inverse significant relationship between IGF-I and both the BNP and P-III-P value in surgically treated acromegaly patients. These observations suggest that appropriate levels of IGF-I have beneficial cardioprotective effects after surgery in patients with acromegaly. (Circ J 2007; 71: 1955 - 1957)

The purpose of this study was to examine the effect of graded conditioning contractions of the antagonist knee flexor muscles on the output characteristics of knee extensor muscles in healthy humans. Eight male university students performed maximum isometric contractions of knee extensors, preceded by isometric conditioning contractions of the antagonist knee flexors. The developed force and electromyographic (EMG) amplitudes of the knee extensors after the conditioning contraction were measured and compared with those of simple knee extension without conditioning. The forces of the conditioning flexor contraction were set at three levels: low (20% of maximum voluntary contraction: MVC), moderate (60% of MVC), and high (100% of MVC). The EMG amplitudes of the vastus medialis, vastus lateralis, and rectus femoris muscle were recorded and the root mean square amplitudes were calculated. The strongest enhancement of the extension force was obtained by moderate intensity conditioning contraction (108.95+/-1.87% of simple knee extension), although high intensity conditioning also induced a significant increase (105.41+/-2.69%). Low intensity conditioning did not cause a significant enhancement of the contraction force (103.17+/-2.99%). Similarly, the EMG amplitudes were significantly increased by moderate and/or high conditioning. These results suggest that antagonist conditioning contraction of moderate intensities is sufficient and may be optimal to potentiate knee extensor contraction.

The human airway trypsin-like protease (HAT) is a respiratory epithelium-associated, type II transmembrane serine protease, which is also detected as an extracellular enzyme in lung fluids during airway inflammatory disorders. We have evaluated its capacity to affect the urokinase-type plasminogen activator receptor (uPAR), a membrane glycolipid-anchored, three-domain (D1D2D3) glycoprotein that plays a crucial role in innate immunity and inflammation by supporting cell migration and matrix degradation, with structure and biological properties that can be regulated via limited endoproteolysis. With the use of immunoblotting, flow immunocytometry, and ELISA analyses applied to a recombinant uPAR protein and to uPAR-expressing monocytic and human bronchial epithelial cells, it was shown that exposure of uPAR to soluble HAT in the range of 10–500 nM resulted in the proteolytic processing of the full-length (D1D2D3) into the truncated (D2D3) species, with cleavage occurring in the D1 to D2 linker sequence after arginine residues at position 83 and 89. Using immunohistochemistry, we found that both HAT and uPAR were expressed in the human bronchial epithelium. Moreover, transient cotransfection in epithelial cells showed that membrane coexpression of the two partners produced a constitutive and extensive shedding of the D1 domain, occurring for membrane-associated HAT concentrations in the nanomolar range. Because the truncated receptor was found to be unable to bind two of the major uPAR ligands, the adhesive matrix protein vitronectin and the serine protease urokinase, it thus appears that proteolytic regulation of uPAR by HAT is likely to modulate cell adherence and motility, as well as tissue remodeling during the inflammatory response in the airways.

Transport properties of P-and Sb-doped Yb4As3 weremeasured. The electrical resistivity measurement of Yb4 (As0. 88Sb0. 12) 3 under various pressure up to 8 GPa was performed These results were compared with that of pure Yb4As3 under high pressure. The substitution effect of Sb is very similar to the pressure effect, however, there is a big differencebetween them. This similarity strongly suggests that the mobility of the electron is much smaller compared to that of the hole.

The Mossbauer spectra have been measured for Sr3Fe2O7-δ at ambient conditions and for Sr3Fe2O6. 80 under pressure. The iron sites are subdivided into two electronically nonequivalent states, that is, one is a tetravalent state and the other a trivalent one. The concentration and the isomer shift of tetravalent site depend on not only the oxygen vacancies but also pressure. It is found that the value of center shift exhibits a discontinuous decrease with increasing pressure and the change of pressure dependence around 8. 5 GPa

By repeatedly introducing hydrophilic polyethylene glycol (PEG) spacer (2) onto affinity resin bearing a bioactive peptide (1/2 secretory leukocyte protease inhibitor, 1/2SLPI) as a ligand, the adsorption of nonspecific binding proteins was effectively reduced and the purification efficacy of elastase, which is one of the target molecules for 1/2SLPI, from a protein mixture was improved. Moreover, using this resin, we also successfully detected L-plastin, as an endogenous target molecule for SLPI, from HL-60 cell lysate.

The low-temperature specific heat and the X-ray diffraction have been measured for Ti1+xS2 (0 < x < 0.1) with the well-defined composition. The obtained lattice and internal parameters increase linearly with the Ti content. The observed electronic specific heat increases linearly with x13. This linear relation changes its slope at x13 = 0.26 and the value goes to zero at x = 0. It is concluded that the electronic structure of stoichiometric TiS2 has a band gap and two Ti-based conduction bands with a small energy difference.

The low-temperature specific heat and Mossbauer spectra have been measured for Sr 3 Fe 2 O 7− y . The iron sites are subdivided into two electronically nonequivalent sites, that is, one is a tetravalent site and the other a trivalent one. The concentration and the isomer shift of tetravalent site depend on the oxygen vacancies. The electronic specific heat of Sr 3 Fe 2 O 6.92 is smaller by one order than those of Sr 3 Fe 2 O 7− y above y = 0.2. It is concluded that the electronic state of Sr 3 Fe 2 O 7− y changes around y ∼ 0.15.

The specific heat at the temperature range from 1.7 to 30 K and the dHvA effect at 0.55 K in the field up to 9.5 T have been measured for Pauli paramagnetic NiAs. The electronic specific heat coefficient is 3.0 mJ/(K2 mol) which is in good agreement with the value estimated from the calculated density of states. It is found that the dHvA oscillation in the field along [0001] consists of four frequencies which correspond to the extremal areas in the k-space of 0.06, 0.11, 0.16 and o.18 A−2.

ESRD is characterized by an interstitial infiltrate of inflammatory cells in association with tubular atrophy, epithelial mesenchymal transdifferentiation (EMT), and interstitial fibrosis. Human proximal tubular epithelial cells (HK2 cells) undergo EMT in response to activated PBMC conditioned medium (aPBMC-CM), showing acquisition of a fibroblastoid morphology, increased fibronectin-EDA (EDA) expression, loss of junctional E-cadherin localization, and cytokeratin 19 (K19) expression. The signaling pathway(s) that regulates EMT in response to aPBMC-CM is not well understood. This study shows that aPBMC-CM induces a rapid activation of RhoA, Rac1, and Cdc42 activity in HK2 cells from 15 min to 48 h. Moreover, infection with adenovirus expressing constitutively active RhoA, Rac1, and Cdc42 significantly increased the expression of EDA and downregulated expression of E-cadherin and K19. Dominant negative RhoA expression suppressed aPBMC-CM-induced upregulation of EDA but did not restore the expression of E-cadherin and K19. Constitutively active RhoA activated the Rho kinase and its downstream effectors, whereas constitutively active Rac1 and Cdc42 activated the P21-activated protein kinase in epithelial cells. In further experiments, HK2 cells were treated with toxin B, exoenzyme C3, Y-27632, and HA1077. These strategies, inhibiting the Rho/Rho kinase pathway, as well as the Rac1/Cdc42/P21-activated protein kinase pathway, blocked transdifferentiation of HK2 cells in response to aPBMC-CM. To conclude, these results indicate that aPBMC-CM activates RhoA, Rac1, and Cdc42 and their downstream effectors, leading to HK2 cells undergoing transdifferentiation. Therefore, activation of small RhoGTPases is a key step in the mechanism of EMT and likely to be a contributor to tubulointerstitial fibrosis.

We have systematically investigated the structural and electrical properties of stoichiometric FeS compound under pressure at low temperature. It is found that two successive first-order pressure-induced structural phase transitions occur at 5.0(2) and 7.0(5) GPa (at 17 K). Furthermore, the pressure dependence of volume changes at around 2.3 GPa. The temperatures dependence of resistivity above 3.0 GPa clearly shows a metallic behavior below 100 K. Consequently, the new isostructural nonmetal-metal transition occurs at around 2.5 GPa in FeS. The transition is almost continuous at 17 K because the volume reduction is less than 0.3% at the transition. In the vicinity of the phase transition at the metallic phase, the pressure dependences of coefficient of quadratic temperature dependence in resistivity and residual resistivity show the critically divergent behaviors.

Background Fibroblastic foci are characteristic features in lung parenchyma of patients with idiopathic pulmonary fibrosis (IPF). They comprise aggregates of mesenchymal cells which underlie sites of unresolved epithelial injury and are associated with progression of fibrosis. However, the cellular origins of these mesenchymal phenotypes remain unclear. We examined whether the potent fibrogenic cytokine TGF-β1 could induce epithelial mesenchymal transition (EMT) in the human alveolar epithelial cell line, A549, and investigated the signaling pathway of TGF-β1-mediated EMT. Methods A549 cells were examined for evidence of EMT after treatment with TGF-β1. EMT was assessed by: morphology under phase-contrast microscopy; Western analysis of cell lysates for expression of mesenchymal phenotypic markers including fibronectin EDA (Fn-EDA), and expression of epithelial phenotypic markers including E-cadherin (E-cad). Markers of fibrogenesis, including collagens and connective tissue growth factor (CTGF) were also evaluated by measuring mRNA level using RT-PCR, and protein by immunofluorescence or Western blotting. Signaling pathways for EMT were characterized by Western analysis of cell lysates using monoclonal antibodies to detect phosphorylated Erk1/2 and Smad2 after TGF-β1 treatment in the presence or absence of MEK inhibitors. The role of Smad2 in TGF-β1-mediated EMT was investigated using siRNA. Results The data showed that TGF-β1, but not TNF-α or IL-1β, induced A549 cells with an alveolar epithelial type II cell phenotype to undergo EMT in a time-and concentration-dependent manner. The process of EMT was accompanied by morphological alteration and expression of the fibroblast phenotypic markers Fn-EDA and vimentin, concomitant with a downregulation of the epithelial phenotype marker E-cad. Furthermore, cells that had undergone EMT showed enhanced expression of markers of fibrogenesis including collagens type I and III and CTGF. MMP-2 expression was also evidenced. TGF-β1-induced EMT occurred through phosphorylation of Smad2 and was inhibited by Smad2 gene silencing; MEK inhibitors failed to attenuate either EMT-associated Smad2 phosphorylation or the observed phenotypic changes. Conclusion Our study shows that TGF-β1 induces A549 alveolar epithelial cells to undergo EMT via Smad2 activation. Our data support the concept of EMT in lung epithelial cells, and suggest the need for further studies to investigate the phenomenon.

The degree of increase BMD (bone mineral density) alone fails to account for the broader effectiveness of vitamin D in reducing the risk of fractures related to osteoporosis. The concept of bone strength also has evolved, integrating those traditional measures of bone quantity (mass) with more recently examined components of bone quality, for example, bone micro-architecture, degree of mineralization, accumulation of micro-damage, and bone turnover. In this review, the effects of vitamin D on bone quality, especially, on bone micro-architecture and degree of mineralization will be discussed.

Six novel chymase inhibitors, SF2809-I, SF2809-II, SF2809-III, SF2809-IV, SF2809-V and SF2809-VI, were isolated from the fermentation broth of an actinomycete strain SF2809. The strain was identified as Dactylosporangium sp. by morphological, chemotaxonomical and phylogenetic studies. These six novel compounds inhibited recombinant human chymase in the range between IC50 of 0.014 and 7.3 microM. However, they showed little or no inhibitory activity against chymotrypsin or cathepsin G, even though these two and chymase belong to the chymotryptic serine protease family. This result indicates that these compounds work as specific chymase inhibitors.