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.     

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).     

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.     

In vivo mutagenesis by Escherichia coli DNA polymerase I. Ile(709) in motif A functions in base selection

The fidelity of DNA replication by Escherichia coli DNA polymerase I (pol I) was assessed in vivo using a reporter plasmid bearing a ColE1-type origin and an ochre codon in the beta-lactamase gene. We screened 53 single mutants within the region Val(700)-Arg(712) in the polymerase active-site motif A. Only replacement of Ile(709) yielded mutator polymerases, with substitution of Met, Asn, Phe, or Ala increasing the beta-lactamase reversion frequency 5-23-fold. Steady-state kinetic analysis of the I709F polymerase revealed reductions in apparent K(m) values for both insertion of non-complementary nucleotides and extension of mispaired primer termini. Abolishment of the 3'-5' exonuclease activity of wild-type pol I increased mutation frequency 4-fold, whereas the combination of I709F and lack of the 3'-5' exonuclease yielded a 400-fold increase. We conclude that accurate discrimination of the incoming nucleotide at the polymerase domain is more critical than exonucleolytic proofreading for the fidelity of pol I in vivo. Surprisingly, the I709F polymerase enhanced mutagenesis in chromosomal DNA, although the increase was 10-fold less than in plasmid DNA. Our findings indicate the feasibility of obtaining desired mutations by replicating a target gene at a specific locus in a plasmid under continuous selection pressure.     

Cell transfer printing from patterned poly(ethylene glycol)-oleyl surfaces to biological hydrogels for rapid and efficient cell micropatterning

Cell transfer printing from patterned poly(ethylene glycol)-oleyl surfaces onto biological hydrogel sheets is investigated herein, as a new cell stamping method for both cell microarray and tissue engineering. By overlaying a hydrogel sheet on the cells immobilized on the poly(ethylene glycol)-oleyl surface and successively peeling it off, the immobilized cells were transferred onto a hydrogel sheet because the adhesive interaction between the cells and the hydrogel was stronger than that between the cells and the poly(ethylene glycol)-oleyl surface. Four types of human cell could be efficiently transferred onto a rigid collagen sheet. The transfer printing ratios, for all cells, were above 80% and achieved within 90 min. A cell microarray was successfully prepared on a collagen gel sheet using the present stamping method. We have also demonstrated that the transferred pattern of endothelial cells is transformed to the patterned tube-like structure on the reconstituted basement membrane matrix. Finally, the patterns of two types of endothelial cell are shown to be easily prepared on the matrix, and the desired tube-like structures, including the orderly pattern of the two different cells, were formed spontaneously. Thus, the present poly(ethylene glycol)-oleyl coated substrates are useful for rapid and efficient cell stamping, in the preparation of multi-cellular pattern on extracellular matrices.     

The Hypoxia-Inducible Epigenetic Regulators Jmjd1a and G9a Provide a Mechanistic Link between Angiogenesis and Tumor Growth

Hypoxia promotes stem cell maintenance and tumor progression, but it remains unclear how it regulates long-term adaptation toward these processes. We reveal a striking downregulation of the hypoxia-inducible histone H3 lysine 9 (H3K9) demethylase JMJD1A as a hallmark of clinical human germ cell-derived tumors, such as seminomas, yolk sac tumors, and embryonal carcinomas. Jmjd1a was not essential for stem cell self-renewal but played a crucial role as a tumor suppressor in opposition to the hypoxia-regulated oncogenic H3K9 methyltransferase G9a. Importantly, loss of Jmjd1a resulted in increased tumor growth, whereas loss of G9a produced smaller tumors. Pharmacological inhibition of G9a also resulted in attenuation of tumor growth, offering a novel therapeutic strategy for germ cell-derived tumors. Finally, Jmjd1a and G9a drive mutually opposing expression of the antiangiogenic factor genes Robo4, Igfbp4, Notch4, and Tfpi accompanied by changes in H3K9 methylation status. Thus, we demonstrate a novel mechanistic link whereby hypoxia-regulated epigenetic changes are instrumental for the control of tumor growth through coordinated dysregulation of antiangiogenic gene expression.     

CpG DNA/zymosan complex to enhance cytokine secretion owing to the cocktail effect

Zymosan, classified among beta-(1-->3)-d-glucans, is produced from the cell wall of yeast and well known to induce proinflammatory cytokines when ingested by immune cells. We found that zymosan forms a complex with immunostimulatory CpG DNA, where both zymosan and CpG DNA can induce cytokine secretion according to the different mechanisms (i.e., recognized by different receptors). The complex activated macrophages and induced cytokine secretion, more efficiently than separate administration of zymosan or CpG DNA. Microscopic observation showed that this increment of the cytokine secretion can be explained by the fact that zymosan and zymosan/CpG DNA complex are up-taken more than naked CpG DNA. Additionally, existence of two different immunostimulants in the same cells may enhance the immunoresponse. This report presents a new strategy to construct a delivering vehicle for CpG DNA and to enhance its activity with the 'cocktail effect' of the two immunostimulants.     

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.