A Highlights from MBoC Selection: p52Shc regulates the sustainability of ERK activation in a RAF-independent manner

p52SHC (SHC) and GRB2 are adaptor proteins involved in the RAS/MAPK (ERK) pathway mediating signals from cell-surface receptors to various cytoplasmic proteins. To further examine their roles in signal transduction, we studied the translocation of fluorescently labeled SHC and GRB2 to the cell surface, caused by the activation of ERBB receptors by heregulin (HRG). We simultaneously evaluated activated ERK translocation to the nucleus. Unexpectedly, the translocation dynamics of SHC were sustained when those of GRB2 were transient. The sustained localization of SHC positively correlated with the sustained nuclear localization of ERK, which became more transient after SHC knockdown. SHC-mediated PI3K activation was required to maintain the sustainability of the ERK translocation regulating MEK but not RAF. In cells overexpressing ERBB1, SHC translocation became transient, and the HRG-induced cell fate shifted from a differentiation to a proliferation bias. Our results indicate that SHC and GRB2 functions are not redundant but that SHC plays the critical role in the temporal regulation of ERK activation.     

Lignin-based electrospun nanofibers reinforced with cellulose nanocrystals

Lignin-based fibers were produced by electrospinning aqueous dispersions of lignin, poly(vinyl alcohol) (PVA), and cellulose nanocrystals (CNCs). Defect-free nanofibers with up to 90 wt % lignin and 15% CNCs were achieved. The properties of the aqueous dispersions, including viscosity, electrical conductivity, and surface tension, were examined and correlated to the electrospinnability and resulting morphology of the composite fibers. A ternary lignin-PVA-water phase diagram was constructed as a tool to rationalize the effect of mixing ratios on the dispersion electrospinability and morphology of the resulting fibers. The influence of reinforcing CNCs on the thermal properties of the multicomponent fibers was investigated by using thermal gravimetric analysis and differential scanning calorimetry. The thermal stability of the system was observed to increase owing to a strong interaction of the lignin-PVA matrix with the dispersed CNCs, mainly via hydrogen bonding, as observed in Fourier transform infrared spectroscopy experiments.     

Production of 2-phenylethanol in roses as the dominant floral scent compound from L-phenylalanine by two key enzymes, a PLP-dependent decarboxylase and a phenylacetaldehyde reductase

We investigated the biosynthetic pathway for 2-phenylethanol, the dominant floral scent compound in roses, using enzyme assays. L-[(2)H8] Phenylalanine was converted to [(2)H8] phenylacetaldehyde and [(2)H8]-2-phenylethanol by two enzymes derived from the flower petals of R. 'Hoh-Jun,' these being identified as pyridoxal-5'-phosphate-dependent L-aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). The activity of rose petal AADC to yield phenylacetaldehyde was nine times higher toward L-phenylalanine than toward its D-isomer, and this conversion was not inhibited by iproniazid, a specific inhibitor of monoamine oxidase. Under aerobic conditions, rose petal AADC stoichiometrically produced NH3 together with phenylacetaldehyde during the course of decarboxylation and oxidation, followed by the hydrolysis of L-phenylalanine. Phenylacetaldehyde was subsequently converted to 2-phenylethanol by the action of PAR. PAR showed specificity toward several volatile aldehydes.     

Type I IFN-mediated enhancement of anti-leukemic cytotoxicity of gammadelta T cells expanded from peripheral blood cells by stimulation with zoledronate

BACKGROUND: In order to establish efficient gammadelta T-cell based tumor immunotherapy, we explored a method to enhance the cytotoxicity of gammadelta T cells against leukemia cells by stimulating gammadelta T cells with type I IFN. METHODS: Gammadelta T cells were expanded from normal PBMC by culturing with zoledronate and a low concentration of IL-2 for 2 weeks. For the activation of gammadelta T cells, gammadelta T cells were cultured with type I IFN (HLBI, IFN-alpha2b and IFN-beta) for 1-3 days. The cytotoxicity of HLBI-activated gammadelta T cells against leukemia cell lines and fresh leukemia cells was evaluated by 51Cr-release assay. RESULTS: Gammadelta T cells, which were expanded and purified with magnetic beads using an anti-gammadelta TCR MAb, were demonstrated to be cytotoxic against leukemia cell lines of both lymphoid and myeloid origin and fresh myeloid leukemia cells. By culturing expanded gammadelta T cells with type I IFN, the expression of the activation marker CD69 was increased and the cytometric bead array showed an elevated production of IFN-gamma by gammadelta T cells. In addition, the cytotoxicity of gammadelta T cells against leukemia cells was definitely enhanced by culturing gammadelta T cells with HLBI. DISCUSSION: The present study has demonstrated that type I IFN could enhance the anti-leukemic cytotoxicity of expanded gammadelta T cells, which implies that in vitro bisphosphonate (such as zoledronate)-expanded and type I IFN-activated gammadelta T cells could be applied to immunotherapy for hematologic malignancies such as leukemia and lymphoma.     

The Identification of a Novel Gene,MAPO2, That Is Involved in the Induction of Apoptosis Triggered by O6-Methylguanine

O⁶-Methylguanine, one of alkylated DNA bases, is especially mutagenic. Cells containing this lesion are eliminated by induction of apoptosis, associated with the function of mismatch repair (MMR) proteins. A retrovirus-mediated gene-trap mutagenesis was used to isolate new genes related to the induction of apoptosis, triggered by the treatment with an alkylating agent, N-methyl-N-nitrosourea (MNU). This report describes the identification of a novel gene, MAPO2 (O⁶-methylguanine-induced apoptosis 2), which is originally annotated as C1orf201. The MAPO2 gene is conserved among a wide variety of multicellular organisms and encodes a protein containing characteristic PxPxxY repeats. To elucidate the function of the gene product in the apoptosis pathway, a human cell line derived from HeLa MR cells, in which the MAPO2 gene was stably knocked down by expressing specific miRNA, was constructed. The knockdown cells grew at the same rate as HeLa MR, thus indicating that MAPO2 played no role in the cellular growth. After exposure to MNU, HeLa MR cells and the knockdown cells underwent cell cycle arrest at G₂/M phase, however, the production of the sub-G₁ population in the knockdown cells was significantly suppressed in comparison to that in HeLa MR cells. Moreover, the activation of BAK and caspase-3, and depolarization of mitochondrial membrane, hallmarks for the induction of apoptosis, were also suppressed in the knockdown cells. These results suggest that the MAPO2 gene product might positively contribute to the induction of apoptosis triggered by O⁶-methylguanine.     

Synthesis of <Emphasis Type="SmallCaps">dl</Emphasis>-tryptophan by modified broad specificity amino acid racemase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IFO 12996

Broad specificity amino acid racemase (E.C. 5.1.1.10) from Pseudomonas putida IFO 12996 (BAR) is a unique racemase because of its broad substrate specificity. BAR has been considered as a possible catalyst which directly converts inexpensive l-amino acids to dl-amino acid racemates. The gene encoding BAR was cloned to utilize BAR for the synthesis of d-amino acids, especially d-Trp which is an important intermediate of pharmaceuticals. The substrate specificity of cloned BAR covered all of the standard amino acids; however, the activity toward Trp was low. Then, we performed random mutagenesis on bar to obtain mutant BAR derivatives with high activity for Trp. Five positive mutants were isolated after the two-step screening of the randomly mutated BAR. After the determination of the amino acid substitutions in these mutants, it was suggested that the substitutions at Y396 and I384 increased the Trp specific racemization activity and the racemization activity for overall amino acids, respectively. Among the positive mutants, I384M mutant BAR showed the highest activity for Trp. l-Trp (20 mM) was successfully racemized, and the proportion of d-Trp was reached 43% using I384M mutant BAR, while wild-type BAR racemized only 6% of initial l-Trp.     

Crystal structure of the RuvA-RuvB complex: a structural basis for the Holliday junction migrating motor machinery

We present the X-ray structure of the RuvA-RuvB complex, which plays a crucial role in ATP-dependent branch migration. Two RuvA tetramers form the symmetric and closed octameric shell, where four RuvA domain IIIs spring out in the two opposite directions to be individually caught by a single RuvB. The binding of domain III deforms the protruding beta hairpin in the N-terminal domain of RuvB and thereby appears to induce a functional and less symmetric RuvB hexameric ring. The model of the RuvA-RuvB junction DNA ternary complex, constructed by fitting the X-ray structure into the averaged electron microscopic images of the RuvA-RuvB junction, appears to be more compatible with the branch migration mode of a fixed RuvA-RuvB interaction than with a rotational interaction mode.