Five Amino Acid Residues Responsible for the High Stability of Hydrogenobacter thermophilus Cytochrome c552

Five amino acid residues responsible for extreme stability have been identified in cytochrome c552 (HT c552) from a thermophilic bacterium, Hydrogenobacter thermophilus. The five residues, which are spatially distributed in three regions of HT c552, were replaced with the corresponding residues in the homologous but less stable cytochrome c551 (PA c551) from Pseudomonas aeruginosa. The quintuple HT c552 variant (A7F/M13V/Y34F/Y43E/I78V) showed the same stability against guanidine hydrochloride denaturation as that of PA c551, suggesting that the five residues in HT c552 necessarily and sufficiently contribute to the overall stability. In the three HT c552 variants carrying mutations in each of the three regions, the Y34F/Y43E mutations resulted in the greatest destabilization, by –13.3 kJ mol–1, followed by A7F/M13V (–3.3 kJ mol–1) and then I78V (–1.5 kJ mol–1). The order of destabilization in HT c552 was the same as that of stabilization in PA c551 with reverse mutations such as F34Y/E43Y, F7A/V13M, and V78I (13.4, 10.3, and 0.3 kJ mol–1, respectively). The results of guanidine hydrochloride denaturation were consistent with those of thermal denaturation for the same variants. The present study established a method for reciprocal mutation analysis. The effects of side-chain contacts were experimentally evaluated by swapping the residues between the two homologous proteins that differ in stability. A comparative study of the two proteins was a useful tool for assessing the amino acid contribution to the overall stability.This work was supported in part by grants from Hiroshima University, the Noda Institute for Scientific Research, and the Japanese Ministry of Education, Science and Culture (grants-in-aid for Scientific Research on Priority Areas)

p90RSK Targets the ERK5-CHIP Ubiquitin E3 Ligase Activity in Diabetic Hearts and Promotes Cardiac Apoptosis and Dysfunction

RATIONALE: Cardiomyocyte apoptosis is one of the key events in the development and progression of heart failure, and a crucial role for ICER (inducible cAMP early repressor) in this process has been previously reported. ERK5 is known to inhibit cardiac apoptosis after myocardial infarction (MI), especially in hyperglycemic states, via association with CHIP ubiquitin (Ub) ligase and subsequent up-regulation of CHIP ligase activity, which induces ICER ubiquitination and subsequent protein degradation. The regulatory mechanism governing ERK5/CHIP interaction is unknown. OBJECTIVE: We previously demonstrated increased p90RSK activation in the diabetic heart. As a logical extension of this work, we now investigate whether p90RSK activation inhibits ERK5-mediated CHIP activation, and subsequently increases ICER levels and apoptosis. METHODS AND RESULTS: p90RSK activation inhibits ERK5/CHIP association and CHIP Ub ligase activity. p90RSK and CHIP share a common binding site in the ERK5 C-terminal domain (aa571-807). Overexpression of either p90RSK or an ERK5 fragment (aa571-807) inhibits ERK5/CHIP association, suggesting that p90RSK and CHIP competes for ERK5 binding and that p90RSK activation is critical for inhibiting ERK5/CHIP interaction. We also identified ERK5-S496 as being directly phosphorylated by p90RSK, and demonstrated that an ERK5-S496A mutant significantly impairs Angiotensin II-mediated inhibition of CHIP activity and subsequent increase in ICER levels. In vivo, either cardiac specific depletion of ERK5 or overexpression of p90RSK inhibits CHIP activity and accelerates cardiac apoptosis after MI--a phenomenon fully reversible by activating ERK5. CONCLUSION: These data suggest a role for p90RSK in inhibiting CHIP activity and promoting cardiac apoptosis through binding to and phosphorylation of ERK5-S496

p90RSK Targets the ERK5-CHIP Ubiquitin E3 Ligase Activity in Diabetic Hearts and Promotes Cardiac Apoptosis and Dysfunction

Cardiomyocyte apoptosis is one of the key events in the development and progression of heart failure, and a crucial role for ICER (inducible cAMP early repressor) in this process has been previously reported. ERK5 is known to inhibit cardiac apoptosis after myocardial infarction (MI), especially in hyperglycemic states, via association with CHIP ubiquitin (Ub) ligase and subsequent up-regulation of CHIP ligase activity, which induces ICER ubiquitination and subsequent protein degradation. The regulatory mechanism governing ERK5/CHIP interaction is unknown

Boundary-only integral equation approach based on polynomial expansion of plasma current profile to solve the Grad–Shafranov equation

A new type of boundary element method (BEM) has been applied to solve the Grad–Shafranov equation and to give a distribution of magnetic flux function in a tokamak nuclear fusion device. The quantity μ0rjphiv related to the plasma current profile is expanded into a two-dimensional polynomial. Using the particular solution of the Grad–Shafranov equation with this inhomogeneous polynomial source and applying Green's second identity, the domain integral related to the plasma current is transformed into an equivalent boundary integral. Domain discretization is not required in this formulation, thus preserving all the advantages of the BEM. Numerical computations of all boundary integrals are only required in the initial stage of the eigenvalue iteration, so that the number of eigenvalue iterations does not hamper the total computing time. Test calculations demonstrated that the present method provides stable and accurate solutions

Biosynthesis of Cyclochlorotine : Identification of the Genes Involved in Oxidative Transformations and Intramolecular O,N-Transacylation

Mycotoxin cyclochlorotine (1) and structurally related astins are cyclic pentapeptides containing unique nonproteinogenic amino acids, such as beta-phenylalanine, L-allo-threonine, and 3,4-dichloroproline. Herein, we report the biosynthetic pathway for 1, which involves intriguing tailoring processes mediated by DUF3328 proteins, including stereo- and regiospecific chlorination and hydroxylation and intramolecular O,N-transacylation. Our findings demonstrate that DUF3328 proteins, which are known to be involved in oxidative cyclization of fungal ribosomal peptides, have much higher functional diversity than previously expected

Ascomycete Aspergillus oryzae Is an Efficient Expression Host for Production of Basidiomycete Terpenes by Using Genomic DNA Sequences

Basidiomycete fungi are an attractive resource for biologically active natural products for use in pharmaceutically relevant compounds. Recently, genome projects on mushroom fungi have provided a great deal of biosynthetic gene cluster information. However, functional analyses of the gene clusters for natural products were largely unexplored because of the difficulty of cDNA preparation and lack of gene manipulation tools for basidiomycete fungi. To develop a versatile host for basidiomycete genes, we examined gene expression using genomic DNA sequences in the robust ascomycete host Aspergillus oryzae, which is frequently used for the production of metabolites from filamentous fungi. Exhaustive expression of 30 terpene synthase genes from the basidiomycetes Clitopilus pseudo-pinsitus and Stereum hirsutum showed two splicing patterns, i.e., completely spliced cDNAs giving terpenes (15 cases) and mostly spliced cDNAs, indicating that A. oryzae correctly spliced most introns at the predicted positions and lengths. The mostly spliced cDNAs were expressed after PCR-based removal of introns, resulting in the successful production of terpenes (14 cases). During this study, we observed relatively frequent mispredictions in the automated program. Hence, the complementary use of A. oryzae expression and automated prediction will be a powerful tool for genome mining. IMPORTANCE The recent large influx of genome sequences from basidiomycetes, which are prolific producers of bioactive natural products, may provide opportunities to develop novel drug candidates. The development of a reliable expression system is essential for the genome mining of natural products because of the lack of a tractable host for heterologous expression of basidiomycete genes. For this purpose, we applied the ascomycete Aspergillus oryzae system for the direct expression of fungal natural product biosynthetic genes from genomic DNA. Using this system, 29 sesquiterpene synthase genes and diterpene biosynthetic genes for bioactive pleuromutilin were successfully expressed. Together with the use of computational tools for intron prediction, this Aspergillus oryzae system represents a practical method for the production of basidiomycete natural products

Preparation of RF-(VM-SiO2)n-RF/AM-Cellu Nanocomposites, and Use Thereof for the Modification of Glass and Filter Paper Surfaces: Creation of a Glass Thermoresponsive Switching Behavior and an Efficient Separation Paper Membrane

Fluoroalkyl end-capped vinyltrimethoxysilane oligomeric silica/alkyl-modified cellulose (AM-Cellu) nanocomposites [RF-(CH2-CHSiO2)n-RF/AM-Cellu; n = 2, 3; RF = CF(CF3)OC3F7] were prepared by the sol-gel reactions of the corresponding oligomer [RF-(CH2-CHSi(OMe)3)n-RF] in the presence of AM-Cellu. The nanocomposites thus obtained were applied to the surface modification of glass to exhibit a highly oleophobic/superhydrophilic characteristic on the modified surface at 20 °C. Interestingly, a temperature dependence of contact angle values of dodecane and water was observed on the modified surface at 20~70 °C, and the dodecane contact angle values were found to decrease with increasing the temperatures from 20 to 70 °C to provide from highly oleophobic to superoleophilic characteristics on the surface. On the other hand, the increase of the water contact angle values was observed with the increase in the temperatures under similar conditions to supply superhydrophilic to superhydrophobic characteristics on the modified surface. The corresponding nanocomposites were also applied to the surface modification of the filter paper under similar conditions to afford a superoleophilic/superhydrophobic characteristic on the surface. It was demonstrated that the modified filter paper is effective for the separation membrane for W/O emulsion to isolate the transparent colorless oil

A case of complete remission of Hodgkin lymphoma confirmed histopathologically by neck dissection

Abstract Background Hodgkin lymphoma (HL) is diagnosed definitively by biopsy, and treatment is based on stage. Owing to the nature of the disease, post‐treatment efficacy is determined mainly by fluorodeoxyglucose‐positron emission tomography/computed tomography, and the efficacy of treatment is not confirmed by histopathology. We report a case of tongue cancer after treatment for HL, in which a post‐treatment lymph node with complete remission was histopathologically confirmed by neck dissection. Case The patient was a 74‐year‐old man who was referred to our hospital for cancer on the right side of his tongue. He had previously undergone chemotherapy for HL involving the right side of his neck and achieved complete remission. Because he had cT3N2cM0 tongue cancer, glossectomy and bilateral neck dissection were performed. Surprisingly, histopathological examination revealed that there was neither metastatic lymph nodes nor lymphoma cells in his right neck. Moreover, there was no lymphatic structure in his remnant lymph nodes. Conclusion This was a rare case in which complete remission of HL was confirmed by histopathological analysis. The absence of lymph node structure and lymphatic flow led to contralateral neck lymph node metastases of tongue cancer