Control of tunnel barriers in multi-wall carbon nanotubes using focused ion beam irradiation.

We have formed tunnel barriers in individual multi-wall carbon nanotubes using the Ga focused ion beam irradiation. The barrier height was estimated by the temperature dependence of the current (Arrhenius plot) and the current-voltage curves (Fowler-Nordheim plot). It is shown that the barrier height has a strong correlation with the barrier resistance that is controlled by the dose. Possible origins for the variation in observed barrier characteristics are discussed. Finally, the single electron transistor with two barriers is demonstrated.

On the dual-cone nature of the conical refraction phenomenon.

In conical refraction (CR), a focused Gaussian input beam passing through a biaxial crystal and parallel to one of the optic axes is transformed into a pair of concentric bright rings split by a dark (Poggendorff) ring at the focal plane. Here, we show the generation of a CR transverse pattern that does not present the Poggendorff fine splitting at the focal plane, i.e., it forms a single light ring. This light ring is generated from a nonhomogeneously polarized input light beam obtained by using a spatially inhomogeneous polarizer that mimics the characteristic CR polarization distribution. This polarizer allows modulating the relative intensity between the two CR light cones in accordance with the recently proposed dual-cone model of the CR phenomenon. We show that the absence of interfering rings at the focal plane is caused by the selection of one of the two CR cones.

Super-Gaussian conical refraction beam.

We demonstrate the transformation of Gaussian input beams into super-Gaussian beams with a quasi flat-top transverse profile by means of the conical refraction phenomenon by adjusting the ratio between the ring radius and the waist radius of the input beam to 0.445. We discuss the beam propagation of the super-Gaussian beam and show that it has a confocal parameter three times larger than the one that would be obtained from a Gaussian beam. The experiments performed with a KGd(WO4)2 biaxial crystal are in good agreement with the theoretical predictions.

Wave-vector and polarization dependence of conical refraction.

We experimentally address the wave-vector and polarization dependence of the internal conical refraction phenomenon by demonstrating that an input light beam of elliptical transverse profile refracts into two beams after passing along one of the optic axes of a biaxial crystal, i.e. it exhibits double refraction instead of refracting conically. Such double refraction is investigated by the independent rotation of a linear polarizer and a cylindrical lens. Expressions to describe the position and the intensity pattern of the refracted beams are presented and applied to predict the intensity pattern for an axicon beam propagating along the optic axis of a biaxial crystal.

Granulocyte colony-stimulating factor (G-CSF) in the mechanism of human ovulation and its clinical usefulness.

In 1980, Espey proposed a famous hypothesis that mammalian ovulation is comparable to an inflammatory reaction and many researches have proved the validity of his hypothesis in the last three decades. For example, interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF)- alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and other inflammatory cytokines presence was proven in the preovulatory follicle. Since granulocyte is the major leukocyte and it plays a very important role during inflammation, the importance of granulocyte and its related cytokine, granulocyte colony-stimulating factor (G-CSF) in the mechanism of human ovulation is easily predictable. G-CSF is one of the hemopoietic cytokines and it has strong positive effects on granulocytes. G-CSF increases the number of granulocytes and it improves the function of granulocytes. In this review, the participation of leukocytes in the ovulation mechanism is demonstrated first. Second, the participation of G-CSF is shown in comparison with the above mentioned cytokines. Finally, since G-CSF has been used for more than 20 years as a medicine without severe side effects in the field of oncology, the clinical application of G-CSF for the treatment of an ovulation disorder, luteinized unruptured follicle (LUF), will be discussed.

Methyl-donor deficiency in adolescence affects memory and epigenetic status in the mouse hippocampus.

DNA methylation is one of the essential factors in the control of gene expression. Alteration of the DNA methylation pattern has been linked to various neurological, behavioral and neurocognitive dysfunctions. Recent studies have pointed out the importance of epigenetics in brain development and functions including learning and memory. Nutrients related to one-carbon metabolism are known to play important roles in the maintenance of genomic DNA methylation. Previous studies have shown that the long-term administration of a diet lacking essential one-carbon nutrients such as methionine, choline and folic acid (methyl donors) caused global DNA hypermethylation in the brain. Therefore, the long-term feeding of a methyl-donor-deficient diet may cause abnormal brain development including learning and memory. To confirm this hypothesis, 3-week-old mice were maintained on a folate-, methionine- and choline-deficient (FMCD) or control (CON) diet for 3 weeks. We found that the methyl-donor deficiency impaired both novel object recognition and fear extinction after 3 weeks of treatment. The FMCD group showed spontaneous recovery of fear that differed from that in CON. In addition, we found decreased Gria1 gene expression and specific CpG hypermethylation of the Gria1 promoter region in the FMCD hippocampus. Our data suggest that a chronic dietary lack of methyl donors in the developmental period affects learning, memory and gene expressions in the hippocampus.

Effects of additives on irreversible inactivation of lysozyme at neutral pH and 100 degrees C.

The mechanism of irreversible inactivation of lysozyme at neutral pH at 100 degrees C, and effects of additives on the inactivation were investigated. The thermoinactivation of lysozyme at neutral pH was caused by intra- and intermolecular disulfide exchange and the production of irreversibly denatured lysozyme, which was destabilized by multiple chemical reactions other than disulfide exchange. In addition, independently, deamidation slightly affected the inactivation by causing a decrease of electrostatic interaction between positive charges of lysozyme and negative charges of the bacterial cell wall. As for the effects of additives on the inactivation, a small amount of copper ion suppressed intra- and intermolecular disulfide exchange by catalyzing air oxidation of heat-induced trace amounts of free thiols, and organic reagents (acetamide, ethanol, and glycerol) changed the mechanism of the inactivation to that under acidic conditions by shifting the pKa values of dissociable residues and also suppressed intermolecular disulfide exchange by decreasing hydrophobic interactions.

Stabilization of lysozyme against irreversible inactivation by suppression of chemical reactions.

The effects of additives on the nonenzymatic deamidation of an Asn residue in a peptide and racemization of Asp and/or Asn in lysozyme were investigated at pH 6 and 100 degrees C. These chemical reactions were accelerated by the addition of phosphate ions. Several salts suppressed the deamidation in the presence of phosphate ions, while the salts did not affect the deamidation in the absence of phosphate ion at pH 6 and 100 degrees C. The results indicated that the effect of the salts was due to the suppression of phosphate catalysis. On the other hand, trifluoroethanol (TFE), which induces the conversion of random coiled polypeptides to secondary structured ones, dramatically suppressed the deamidation of an Asn residue in a peptide. The rate of deamidation in the presence of TFE was comparable to that of asparagine (free amino acid), which was very slowly deamidated. Because TFE could not suppress the deamidation of free asparagine, the suppression of the deamidation of an Asn residue in a peptide was attributed to suppression of the catalysis by the peptide bond in the carboxyl terminus. Since the inactivation of lysozyme was caused by multiple chemical reactions such as the deamidation and racemization, it was expected that the inactivation of lysozyme could be prevented by the addition of salts or TFE. Thus, it was confirmed that salts and TFE suppressed the lysozyme inactivation at pH 6 and 100 degrees C.

Association of elastin glycation and calcium deposit in diabetic rat aorta.

The relationship between glycation of the aortic elastin and calcium deposits in the aorta was studied in streptozocin (STZ)-induced diabetic rats. 5-Hydroxymethylfurfural (5-HMF) which was released from aortic elastin by acid, was assayed after STZ treatment as an index of early stage glycation. The amount of released 5-HMF increased at 5 weeks and paradoxically decreased at 10 weeks after STZ treatment, though it remained higher than that of control rats. This paradoxical pattern was reproduced by the in vitro incubation of elastin with glucose and it is presumably due to further advancement of glycation reactions in diabetic rats. The level of 5-HMF did not change significantly in control rats at corresponding time points of 9, 11 and 16 weeks of age. Fluorescence of porcine pancreatic elastase I-digested elastin which served as an index of advanced glycation, increased by 1.6 times at 3 weeks and reached a maximum of 1.9-fold higher than that of control rats at 10 weeks. The calcium content of the aorta at 10 weeks in diabetic rats was significantly increased by 1.4-fold compared with control rats. This study showed that the increased elastin glycation in the aorta even at the early stage of diabetes is associated with calcium deposit in the aorta. These results are consistent with the interpretation that elastin glycation in the aorta is the potential accelerating factor for diabetic macroangiopathy.

[Establishment and characterization of human cholaginocarcinoma, MEC, producing carbohydrate antigen 19-9].

A new tumor cell line MEC was established from pleural effusion of a patient of cholaginocarcinoma. In tissue culture, the cell line grew in the sheet of variant cells and showed the epithelial-like pattern. Histologically, the cell line almost showed the same pattern as those in bile and preural effusion from the patient. Electron microscopic observation of this cell line showed the irregular microvilli on the surface of the cell and the desmosome between cells. The doubling time of the cell line was 40.8 hours. Chromosome counts ranged from 61 to 86. The cell line had 9 marker chromosomes and some variant chromosomes. The cell line was transplanted into the subcutaneous of nude mice and formed the tumor. It showed the moderately differentiated tubular adenocarcinoma the same pattern as the primary tumor. We have recognized the producing and releasing of CA19-9 in the serum from the tumor bearing nude mouse and supernate of the medium as the serum from the patient. The presentation of CA19-9 in the cytosol of the cell line and the tumor cells of nude mouse was recognized in Avidin-Biotin-Peroxidase Complex in immunoloperoxidase techniques. The cell line can grow in serum-free medium. On September, 1990, the cell line has been maintained from 70 passages during about 800 days.

Biological characterization of a novel class of toll-like receptor 7 agonists designed to have reduced systemic activity.

BACKGROUND AND PURPOSE Toll-like receptor 7 (TLR7) agonists have potential in the treatment of allergic diseases. However, the therapeutic utility of current low molecular weight TLR7 agonists is limited by their systemic activity, resulting in unwanted side effects. We have developed a series of TLR7-selective 'antedrugs', including SM-324405 and AZ12441970, which contain an ester group rapidly cleaved in plasma to reduce systemic exposure. EXPERIMENTAL APPROACH Agonist activity at TLR7 of the parent ester and acid metabolite was assessed in vitro in reporter cells and primary cells from a number of species. Pharmacokinetics following a dose to the lungs was assessed in mice and efficacy evaluated in vivo with a mouse allergic airway model. KEY RESULTS Compounds were selective agonists for TLR7 with no crossover to TLR8 and were metabolically unstable in plasma with the acid metabolite showing substantially reduced activity in a number of assays. The compounds inhibited IL-5 production and induced IFN-α, which mediated the inhibition of IL-5. When dosed into the lung the compounds were rapidly metabolized and short-term exposure of the 'antedrug' was sufficient to activate the IFN pathway. AZ12441970 showed efficacy in a mouse allergic airway model with minimal induction of systemic IFN-α, consistent with the low plasma levels of compound. CONCLUSIONS AND IMPLICATIONS The biological and metabolic profiles of these TLR7-selective agonist 'antedrug' compounds are consistent with a new class of compound that could be administered locally for the treatment of allergic diseases, while reducing the risk of systemic side effects. LINKED ARTICLE This article is commented on by Kaufman and Jacoby, pp. 569-572 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01758.x.

The mechanism of irreversible inactivation of lysozyme at pH 4 and 100 degrees C.

The mechanism of irreversible inactivation of lysozyme at pH 4, 100 degrees C, was investigated. It was elucidated that the inactivation was caused by production of molecules in an irreversibly denatured state. From analyses of the mechanism of production of the inactive enzyme, the inactivation was not evoked by a single chemical reaction. The free energy change between the folded and unfolded states decreased by the accumulation of chemical reactions (isomerization of Asp-Gly, deamidation of Asn, racemization of Asp and Asn, and cleavage of the Asp-X-peptide bond) induced at high temperature. Thus, certain molecules were ultimately in the unfolded state even at low temperature and lost activity. Moreover, a good correlation between the stability (free energy change) and the averaged number of chemical reactions that leads to the inactivation was obtained on the basis of some assumptions.

Isolation and characterization of 101-succinimide lysozyme that possesses the cyclic imide at Asp101-Gly102.

Lytic activity of lysozyme solution gradually increased on incubation at pH 4, 40 degrees C. When the solution was analyzed by use of cation-exchange HPLC at pH 5, a new peak appeared with increased incubation time. The derivative in the new peak was identified to be 101-succinimide lysozyme in which cyclic imide formed at Asp101-Gly102. The formation of 101-succinimide lysozyme increased with increases in concentration of acetate buffer. Kinetic analysis of the formation of 101-succinimide lysozyme indicated that the cyclic imide was stable below pH 5 due to suppression of the hydrolysis of cyclic imide. Its lytic activity against M. luteus, which has a negative charge, was 165% at pH 7, whereas its activity against glycol chitin, which has no charge, was 90%. Since the lytic activity of Asn101 lysozyme, where one negative charge is eliminated, reached a maximum of 125%, it was suggested that the increase of lytic activity against bacterial cells in 101-succinimide lysozyme was due not only to the disappearance of the negative charge at Asp101 but also to the removal of steric hindrance at the upper part of the active site cleft.

Stabilization of lysozyme against irreversible inactivation by alterations of the Asp-Gly sequences.

Site-directed mutagenesis was performed at Asp-Gly (48-49, 66-67, 101-102) and Asn-Gly (103-104) sequences of hen egg-white lysozyme to protect the enzyme against irreversible thermoinactivation. Because the lysozyme inactivation was caused by the accumulation of multiple chemical reactions, including the isomerization of the Asp-Gly sequence and the deamidation of Asn [Tomizawa et al. (1994) Biochemistry, 33, 13032-13037], the suppression of these reactions by the substitution of Gly to Ala, or the introduction of a sequence of human-type lysozyme, was attempted and the mutants (where each or all labile sequences were replaced) were prepared. The substitution resulted in the reversible destabilization from 1 to 2 kcal/mol per substitution. The destabilization was caused by the introduction of beta-carbon to the constrained position that had conformational angles within the allowed range for the Gly residue. Despite the decrease in the reversible conformational stability, the mutants had more resistance to irreversible inactivation at pH 4 and 100 degrees C. In particular, the rate of irreversible inactivation of the mutant, which was replaced at four chemically labile sequences, was the latest and corresponded to approximately 18 kcal/mol of the reversible conformational stability. Therefore, replacement of the chemically labile sequence was found to be more effective at protecting enzymes against irreversible thermoinactivation than at strengthening reversible conformational stability.

Antibacterial activity of AM-1155 against penicillin-resistant Streptococcus pneumoniae.

AM-1155, a novel fluoroquinolone, exhibited potent activity against Streptococcus pneumoniae, including penicillin-resistant strains; the MIC90 for 48 clinical isolates was 0.25 mg/L. The antibacterial activity of AM-1155 against S. pneumoniae was higher than that of levofloxacin (MIC90 1 mg/L) and comparable with that of sparfloxacin. The MIC90s of penicillin G and erythromycin were 2 and > 4 mg/L, respectively. AM-1155 showed no cross-resistance to penicillin or erythromycin. For experimental murine pneumonia with S. pneumoniae intermediately resistant to penicillin, oral administration of AM-1155 showed efficacy higher than that of levofloxacin and equal to that of sparfloxacin. The efficacy of AM-1155 was also equal to that of subcutaneous penicillin G administration at the same dosage.

Immobilized ferredoxins for affinity chromatography of ferredoxin-dependent enzymes.

An immobilized ferredoxin more stable than the conventional immobilized spinach ferrodoxin was prepared by reacting CNBr-Sepharose with ferredoxins isolated from barley and Synechococcus vulcanus, a thermophilic blue-green alga. The dissociation constants of immobilized ferredoxin from spinach, barley and S. vulcanus for spinach ferredoxin-NADP reductase were 0.922, 2.505 and 5.209 microM, respectively, whereas those for barley ferredoxin-NADP reductase were 1.159, 0.579 and 2.851 microM, respectively. The order of stability was S. vulcanus greater than barley greater than spinach. The immobilized ferredoxin was applied to the simultaneous detection of ferredoxin-dependent enzymes in spinach chloroplasts. Over 20 polypeptides were detected. Synechococcus ferredoxin could also be immobilized on a Toyopearl gel and repeatedly used in an automated high-performance liquid chromatographic system.