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We addressed the role of nitric oxide (NO) in orexin neuron degeneration that has been observed under various pathological conditions. Administration of an NO donor NOCI8 (50 nmol) into the third ventricle of mice resulted in a significant decrease of orexin -immunoreactive (-IR) neurons, in contrast to a modest change in melanin-concentrating hormone-IR neurons. In addition, NOC18 promoted formation of orexin-A-IR aggregates within orexin neurons. An endoplasmic reticulum stress inducer tunicamycin replicated the effect of NOC18 with regard to decrease of orexin-IR neurons and formation of aggregates. We also found that NOC18 caused an increase in S-nitrosation of protein disulfide isomerase (PDI) and a decrease in PDI activity in hypothalamic tissues. Moreover, PDI inhibitors, such as cystamine and securinine, caused a selective decrease of orexin neurons and promoted formation of orexin-A-IR aggregates. Aggregate formation in orexin-IR neurons was also induced by local injection of small interfering RNA targeting PDI. Interestingly, sleep deprivation for 7 consecutive days induced a selective decrease of orexin-IR neurons, which was preceded by aggre-gate formation in orexin-IR neurons and an increase in S-nitrosated PDI in the hypothalamus. Activity of neuronal NO synthase (nNOS)-positive neurons in the lateral hypothalamus as assessed by c-Fos expression was elevated in response to sleep deprivation. Finally, sleep deprivation-induced decrease of orexin-IR neurons, formation of aggregates, and S-nitrosation of PDI were not observed in nNOS knock-out mice. These results indicate that nNOS-derived NO may mediate specific pathological events in orexin neurons, including neuropeptide misfolding via S-nitrosation and inactivation of PDI. [ABSTRACT FROM AUTHOR]

4 Z,15 Z-bilirubin-IXα (4 Z,15 Z-BR), an endogenous compound that is sparingly soluble in water, binds human serum albumin (HSA) with high affinity in a flexible manner. A phage library displaying recombinant HSA domain II was constructed, after three rounds of panning against immobilized 4 Z,15 Z-BR, and eight clones with high affinity for the pigment were found to contain conserved basic residues, such as lysine or arginine, at positions 195 and 199. The wild type and two mutants, K195A and K199A, of whole HSA as well as stand-alone domain II were expressed in Pichia pastoris for ligand-binding studies. The binding of 4 Z,15 Z-BR to the K195A and K199A mutants was decreased in both whole HSA and the domain II proteins. The P-helicity conformer (P-form) of 4 Z,15 Z-BR was found to preferentially bind to the wild types and the K195A mutants, whereas the M-form bound to the K199A mutants. Photoconversion experiments showed that the P-form of 4 Z,15 Z-BR was transformed into highly water-soluble isomers at a much faster rate than the M-form. In addition, the M-form of 4 Z,15 Z-BR showed higher affinity for domain I than for domain II. The present findings suggest that, whereas both Lys195 and Lys199 in subdomain IIA are important for the high-affinity binding of 4 Z,15 Z-BR, Lys199 plays a more prominent role in the elimination of 4 Z,15 Z-BR. Database [ABSTRACT FROM AUTHOR]

The cyclobutane pyrimidine dimer (CPD) is a major type of DNA damage induced by ultraviolet B (UVB) radiation. CPD photolyase, which absorbs blue/UVA light as an energy source to monomerize dimers, is a crucial factor for determining the sensitivity of rice (Oryza sativa) to UVB radiation. Here, we purified native class II CPD photolyase from rice leaves. As the final purification step, CPD photolyase was bound to CPD-containmg DNA conjugated to magnetic beads and then released by blue-light irradiation. The final purified fraction contained 54- and 56-kD proteins, whereas rice CPD photolyase expressed from Escherichia co/i was a single 55-kD protein. Western-blot analysis using anti-rice CPD photolyase antiserum suggested that both the 54- and 56-kD proteins were the CPD photolyase. Treatment with protein phosphatase revealed that the 56-kD native rice CPD photolyase was phosphorylated, whereas the E. co/i-expressed rice CPD photolyase was not. The purified native rice CPD photolyase also had significantly higher CPD photorepair activity than the E. co/i-expressed CPD photolyase. According to the absorption, emission, and excitation spectra, the purified native rice CPD photolyase possesses both a pterin-like chro- mophore and an FAD chromophore. The binding activity of the native rice CPD photolyase to thymine dimers was higher than that of the E. co/i-expressed CPD photolyase. These results suggest that the structure of the native rice CPD photolyase differs significantly from that of the E. co/i-expressed rice CPD photolyase, and the structural modification of the native CPD photolyase leads to higher activity in rice. [ABSTRACT FROM AUTHOR]

Flap endonuclease 1 (FEN1) is a structure-specific nuclease that removes the RNA/DNA primer associated with Okazaki fragments in DNA replication. Here, crystals of the complex between the catalytic domain of human FEN1 and a DNA product have been obtained. For efficient crystallization screening, a DNA--protein complex crystallization screening (DPCS) kit was designed based on commercial crystallization kits. The crystal was found to belong to space group P21, with unit-cell parameters a = 61.0, b = 101.3, c = 106.4 Å, β = 106.4°. The asymmetric unit is predicted to contain two complexes in the crystallographic asymmetric unit. A diffraction data set was collected to a resolution of 2.75 Å. [ABSTRACT FROM AUTHOR]

The present study was designed to explore whether maternal uninephrectomy affects development of the collecting ducts in fetal kidney. Localization of DNA fragmented cells in the kidney of fetal rats from uninephrectomized mothers were examined by the terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end labeling (TUNEL) method. Immunohistochemistry was used to examine the localizations of bcl-2 gene products. The gene expressions for bcl-2, p53, and WT1 mRNAs were examined by using the semi-quantitative reverse transcript-polymerase chain reaction. TUNEL positive cells were more numerous in the medullary collecting ducts of the fetuses from uninephrectomized mothers than in those of the fetuses from sham-operated ones. The expressions of bcl-2, p53, and WT1 mRNAs were lower in the fetuses from uninephrectomized mothers than in the fetuses from sham-operated ones. Cells in the medullary collecting ducts showed positive reactions to anti- bcl-2 gene products antibody with the reactions being weaker in the fetuses from uninephrectomized mothers. These results showed that maternal uninephrectomy accelerated the development of fetal rat kidney and it was associated with the lowered the expression of bcl-2 in fetal rat kidney. [ABSTRACT FROM AUTHOR]

Flap endonuclease-1 (FEN1) is a key enzyme for maintaining genomic stability and replication. Proliferating cell nuclear antigen (PCNA) binds FEN1 and stimulates its endonuclease activity. The structural basis of the FEN1-PCNA interaction was revealed by the crystal structure of the complex between human FEN1 and PCNA. The main interface involves the C-terminal tail of FEN1, which forms twoß-strands connected by a short helix, theßA-aA-ßB motif, participating inß-ßand hydrophobic interactions with PCNA. These interactions are similar to those previously observed for the p21CIP1/WAF1 peptide. However, this structure involving the full-length enzyme has revealed additional interfaces that are involved in the core domain. The interactions at the interfaces maintain the enzyme in an inactive‘locked-down’orientation and might be utilized in rapid DNA-tracking by preserving the central hole of PCNA for sliding along the DNA. A hinge region present between the core domain and the C-terminal tail of FEN1 would play a role in switching the FEN1 orientation from an inactive to an active orientation. [ABSTRACT FROM AUTHOR]

The input-output characteristic of an ultrasonic motor (USM) has nonlinear elements and changes with a temperature rise or fluctuation of load-mass. Therefore, it is difficult to accomplish satisfactory control performance by using conventional PID controllers. In this paper, we propose a PID controller combined with a neural network (NN-PID controller). In this design method, the controller gains consist of constant gains of the PID controller and variable gains of the NN controller. The weights of the NN are adjusted by the backpropagation method so that the control error can be minimized. This method does not require long learning time of the NN. The effectiveness of the proposed design method is confirmed by experiments using an existing USM. © 2003 Wiley Periodicals, Inc. Electr Eng Jpn, 146(3): 46–54, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/eej.10199 [ABSTRACT FROM AUTHOR]

To ensure the stability of caverns and optimize the support systems, observational design and construction procedures have been used recently for excavating large underground caverns, such as those for pumped-storage power plants, which spontaneously process data monitored during construction and real-time feedback of the results to the next construction steps. In observational design and construction procedures, the cavern support systems should be optimized by monitoring the loosening behavior and correctly predicting and analyzing the subsequent behavior of the cavern. This paper describes an evaluation of loosened zones based on monitored stress of rock and an application of prediction analysis for observational construction techniques. [Copyright &y& Elsevier]

Abstract Background: Proliferating cell nuclear antigen (PCNA) is a ring-shaped protein known as a processivity factor of DNA polymerase δ. In addition to this role, PCNA interacts with a number of other proteins to increase their local concentration at replicated DNA sites. DNA cytosine methyltransferase 1 (Dnmt1), which preserves epigenetic signals by completing the methylation of hemimethylated DNA after DNA replication, has been indicated as one of these PCNA binding proteins by a previous work. However, the molecular mechanisms and functional significance of their association have not yet been studied. Results: Dnmt1 can be readily isolated from nuclear extracts by PCNA affinity chromatography. Studies of the interactions between the two proteins demonstrate that the N-terminal region of Dnmt1, which contains a typical PCNA binding motif, has core PCNA binding activity, and that the remaining portion of the protein exerts a negative influence on the interaction of Dnmt1 with PCNA. The affinity of Dnmt1 for DNA is much higher for DNA bound by PCNA than for free DNA. Furthermore, DNA methylation assays with hemimethylated DNA as a substrate revealed that PCNA clamp-bound DNA is methylated more efficiently by Dnmt1 than is free DNA. Conclusion: These results provide the first biochemical evidence that physical interactions between PCNA and Dnmt1 facilitate the methylation of newly neplicated DNA, on which PCNA remains associated as a functional clamp. [ABSTRACT FROM AUTHOR]

Presents a study which focused on two monoclonal antibodies, 64M3 and 64M5, that share the same binding specificity but differ in their affinities. How the photolesions in DNA were detected and quantitated; Use of single-chain Fv (scFv) derivatives; Materials and methods.

The preparation and analysis of a mutant ribonuclease (RNase) T1 which possesses higher nucleolytic activity than the wild-type enzyme ate described. The gene for the mutant RNase T1 (Tyr45 → Trp45), in which a single amino acid at the binding site of the guanine base has been changed, was constructed by the cassette mutangenesis method using a chemically synthesized gene [Ilkehara, M. et al. (1986) Proc. Natl Acad. Sci. USA 83, 4695–4699]. In order to reduce the nucleolytic activity of the enzyme in vivo, this gene was expressed in Escherichia coli as a fused protein connected through methionine residues to other proteins at both the N- and C-termini. After liberation from the fused protein by cleavage with cyanogen bromide at the methionine junctions, the mutant RNase T1 was purified by column chromatography. The nucleolytic activity toward pGpC increased to 120% of that of wild-type RNase T1. The kinetic parameters of the mutant enzyme demonstrate that this higher nucleolytic activity is due to a higher affinity for the substrate, probably because of an increased slacking effect in the binding pocket for the guanine base. This mutant enzyme also possessed a higher nucleolytic activity against pApC than wild-type RNase T1. [ABSTRACT FROM AUTHOR]