The effects of reactive oxygen species on amphibian aging

To clarify the role of reactive oxygen species (ROS) in the aging process of amphibians, antioxidant enzyme activity and indexes of ROS damage were investigated biochemically using the livers of 3- and 10-year-old Rana nigromaculata frog males and females. Findings revealed no significant difference in survival rate between males and females. Antioxidant enzyme activity displayed an age-related decline. Superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activity in 10-year-old liver decreased 40-80% from 3-year-old liver levels. In contrast, urate oxidase activity in the 10-year-old liver increased more than 200% from 3-year-old liver levels. At the same time levels of ROS damage, including the concentration of inorganic peroxide and thiobarbituric acid reactive substances (TBARS), greatly increased with age. Liver catalase from 10-year-old frogs proved to be more susceptible to aminotriazole and urea, losing approximately 80% of its original activity after 30 min of treatment. It seems likely that liver catalase in older frogs has diverged from liver catalase in younger frogs through oxidative modification. These findings suggest that a decrease in the activity of antioxidant enzymes over time results in increased levels of ROS damage in the livers of older frogs.     

Identification of biologically active sequences in the laminin alpha 4 chain G domain

Laminins are a family of trimeric extracellular matrix proteins consisting of alpha, beta, and gamma chains. So far five different laminin alpha chains have been identified. The laminin alpha 4 chain, which is present in laminin-8/9, is expressed in cells of mesenchymal origin, such as endothelial cells and adipocytes. Previously, we identified heparin-binding sites in the C-terminal globular domain (G domain) of the laminin alpha 4 chain. Here we have focused on the biological functions of the laminin alpha 4 chain G domain and screened active sites using a recombinant protein and synthetic peptides. The rec-alpha 4G protein, comprising the entire G domain, promoted cell attachment activity. The cell attachment activity of rec-alpha 4G was completely blocked by heparin and partially inhibited by EDTA. We synthesized 116 overlapping peptides covering the entire G domain and tested their cell attachment activity. Twenty peptides showed cell attachment activity, and 16 bound to heparin. We further tested the effect of the 20 active peptides in competition assays for cell attachment and heparin binding to rec-alpha 4G protein. A4G6 (LAIKNDNLVYVY), A4G20 (DVISLYNFKHIY), A4G82 (TLFLAHGRLVFM), and A4G83 (LVFMFNVGHKKL), which promoted cell attachment and heparin binding, significantly inhibited both cell attachment and heparin binding to rec-alpha 4G. These results suggest that the four active sites are involved in the biological functions of the laminin alpha 4 chain G domain. Furthermore, rec-alpha 4G, A4G6, and A4G20 were found to interact with syndecan-4. These active peptides may be useful for defining of the molecular mechanism laminin-receptor interactions and laminin-mediated cellular signaling pathways.     

The KDEL receptor modulates the endoplasmic reticulum stress response through mitogen-activated protein kinase signaling cascades

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) evokes the ER stress response. The resultant outcomes are cytoprotective but also proapoptotic. ER chaperones and misfolded proteins exit to the secretory pathway and are retrieved to the ER, during which process the KDEL receptor plays a significant role. Using an expression of a mutant KDEL receptor that lacks the ability for ligand recognition, we show that the impairment of retrieval by the KDEL receptor led to a mis-sorting of the immunoglobulin-binding protein BiP, an ER chaperone that has a retrieval signal from the early secretory pathway, which induced intense ER stress response and an increase in susceptibility to ER stress in HeLa cells. Furthermore, we show that the ER stress response accompanied the activation of p38 mitogen-activated protein (MAP) kinases and c-Jun amino-terminal kinases (JNKs) and that the expression of the mutant KDEL receptor suppressed the activation of p38 and JNK1 but not JNK2. The effect of the expression of the mutant KDEL receptor was consistent with the effect of a specific inhibitor for p38 MAP kinases, because the inhibitor sensitized HeLa cells to ER stress. We also found that activation of the KDEL receptor by the ligand induced the phosphorylation of p38 MAP kinases. These results indicate that the KDEL receptor participates in the ER stress response not only by its retrieval ability but also by modulating MAP kinase signaling, which may affect the outcomes of the mammalian ER stress response.     

Heat shock protein 70 expression induces antitumor immunity during intracellular hyperthermia using magnetite nanoparticles

In this study we demonstrated that heat shock protein (HSP) 70 expression by hyperthermia induced antitumor immunity in the T-9 rat glioma. Our hyperthermic system using magnetic nanoparticles induced necrotic cell death that correlated with HSP70 expression. We purified the HSP70-peptide complexes from the tumor after hyperthermia to investigate whether HSP70 was involved in the antitumor immunity, and we found that in the F344 rats immunized with T-9-derived HSP70 the tumor growth of T-9 was significantly suppressed. Tumor rejection assay after hyperthermic treatment of implanted T-9 cells with incorporated magnetite cationic liposomes (MCL) was performed to investigate whether antitumor immunity was induced by release of HSP70 from the necrotic cells in the F344 rat. Tumor growth was strongly suppressed in the rats subjected to hyperthermia of implanted T-9 cells, and 50% of rats were protected from challenge with T-9 cells. Immunogenicity was enhanced when the HSP70-overexpressing T-9 cells were killed via necrosis in rats by hyperthermia, after which all rats were completely protected from challenge with T-9 cells. Our hyperthermic system produces vaccination with HSP70-peptide via necrotic tumor cell death in vivo, resulting in antitumor immunity. This phenomenon, which may be termed in situ vaccination, has important implications for the development of novel antitumor therapies.     

In vitro and in vivo analyses of a Phe/Tyr switch controlling product specificity of histone lysine methyltransferases

The functional significance of mono-, di-, and tri-methylation of lysine residues within histone proteins is under investigation. Evidence from several model organisms suggests that different methylated states of H3 Lys(9) (H3K9) are generated by specific histone methyltransferases (MTases) to mark distinct types of silent chromatin. Sequence alignment of all histone lysine MTases with known product specificity suggested that a key residue in the active site determines how many methyl groups they add. We examined this possibility both in vitro and in vivo and found that a Phe at the position equivalent to Phe(281) of Neurospora crassa DIM-5 or Phe(1205) of human G9a allows the enzyme to perform di and tri-methylation, whereas a Tyr at this position is restrictive, inhibiting tri-methylation and thus yielding a mono- or di-MTase. Phe to Tyr mutants of both DIM-5 and G9a restrict product specificity in vitro and in vivo without compromising overall catalysis. These mutants were employed to probe the biological significance of mono-, di-, and tri-methylation of H3K9 in both mouse embryonic stem cells and N. crassa. G9a F1205Y, when expressed in G9a (-/-) embryonic stem cells, rescued only H3K9 mono-methylation, but not di-methylation, to wild-type levels yet silenced Mage-a gene expression. When expressed in dim-5 strains, DIM-5 F281Y generated significant levels of mono- and di-H3K9 methylation (which are not observed in wild type Neurospora) as well as tri-methyl H3K9. The altered DIM-5 rescued the growth defect characteristic of dim-5 N. crassa but did not fully rescue the gross DNA hypomethylation of dim-5 strains.     

Low Mw sulfated curdlan with improved water solubility forms macromolecular complexes with polycytidylic acid

The water solubility of curdlan was enhanced by partial sulfation at O-6. Circular dichroism measurements suggest that the sulfated curdlan with the degree of substitution (DS) from 0 to 8.7 mol% forms macromolecular complexes with polycytidylic acid (poly(C)). Although the thermal stability of the complexes decreased with increase in DS, this could be overlapped by addition of NaCl in the concentration above that of serum. The results clearly indicate that the drawback arising from the electrostatic repulsion between the anionic charges can be partially compensated by the presence of salt. Furthermore, the polynucleotide chain complexed with the sulfated curdlan was protected from the enzymatic hydrolysis, corroborating the assumption that the sulfated curdlan has an ability to bind poly(C).     

Rod-like architecture and helicity of the poly(C)/schizophyllan complex observed by AFM and SEM

Microscopic studies of the complex between poly(C) and schizophyllan (SPG), employing both AFM and SEM, revealed that the complex takes the same rod-like architecture on the mica surface as those of the renatured SPG and the original triple helix of SPG, indicating that the complex also has a helical structure. The SEM observations showed the helical pattern on the rod surface, only when the sample was metal shadowed. The pitch evaluated from the image is comparable with that obtained from crystallographic data. The ability to visualize the helical structure can be explained from the hypothesis that the platinum grains may assemble on the sample using the molecular surface of the SPG (or complex) as the template.     

Molecular design of synthetic receptors with dynamic, imprinting, and allosteric functions

Sugar recognition in an aqueous system has been achieved using a boronic acid-diol interaction. Combination with an intramolecular amino group has enabled us to read out the binding process as a change in the fluorescence intensity. The novel interaction has been extended to dynamic sugar sensing utilizing an allosteric effect, molecular imprinting, and control of molecular assemblies.     

Phase separation in the mixture of schizophyllan and poly(ethylene oxide) in aqueous solution driven by a specific interaction between the glucose side chain and poly(ethylene oxide)

We found that the mixture of schizophyllan and poly(ethylene oxide) in aqueous solution underwent phase separation at around 3-4 degrees C, and this temperature was independent of both polymer concentration and the difference in poly(ethylene oxide) molecular weight (Mw 6000 and 70,000). The phase-separation took place at the same temperature at which the optical rotation changed. Since the optical rotation change is ascribed to the difference in the nature of hydrogen bonding between the schizophyllan side chain and water, the phase separation is also considered to be due to an interaction between poly(ethylene oxide) and schizophyllan. The phase-separation temperature increased on changing H2O to D2O in accordance with a change in the optical rotation, confirming the specific interaction essential for the phase separation.     

Further evidence for the gelation ability-structure correlation in sugar-based gelators

Eight methyl glycosides of 4,6-O-benzylidene derivatives of the monosaccharides D-glucose, D-mannose, D-allose and D-altrose were synthesized to systematically study the effect of small configurational changes on the ability to gelate organic solvents. Among the beta anomers, only the D-mannose glycoside exhibits a strong gelation ability, whereas in the alpha-series the D-glucose and D-mannose derivatives act as versatile gelators. Also, as a general rule we found that the beta anomers possess a higher ability to gelate solvents than the alpha anomers. The gelation properties are discussed on the basis of SAXS, FTIR, differential scanning calorimetric (DSC) measurements and scanning electron microscopy (SEM) observations. The temperature-dependent SAXS measurements were carried out to elucidate the sol-gel transition temperature. The present study emphasizes that the saccharide family provides, not only valuable information of the structural requirements for the design of new gelators, but also for molecular assembly systems in general.