Cutting edge: tyk2 is required for the induction and nuclear translocation of Daxx which regulates IFN-alpha-induced suppression of B lymphocyte formation

IFN-alpha inhibits B lymphocyte development, and the nuclear protein Daxx has been reported to be essential for this biological activity. We show in this study that IFN-alpha inhibits the clonal proliferation of B lymphocyte progenitors in response to IL-7 in wild-type, but not in tyk2-deficient, mice. In addition, the IFN-alpha-induced up-regulation and nuclear translocation of Daxx are completely abrogated in the absence of tyk2. Therefore, tyk2 is directly involved in IFN-alpha signaling for the induction and translocation of Daxx, which may result in B lymphocyte growth arrest and/or apoptosis.     

Studies on the formation mechanism and the structure of the anisotropic collagen gel prepared by dialysis-induced anisotropic gelation

We have found that dialysis of 5 mg/mL collagen solution into the phosphate solution with a pH of 7.1 and an ionic strength of 151 mM [corrected] at 25 °C results in a collagen gel with a birefringence and tubular pores aligned parallel to the growth direction of the gel. The time course of averaged diameter of tubular pores during the anisotropic gelation was expressed by a power law with an exponent of 1/3, suggesting that the formation of tubular pores is attributed to a spinodal decomposition-like phase separation. Small angle light scattering patterns and high resolution confocal laser scanning microscope images of the anisotropic collagen gel suggested that the collagen fibrils are aligned perpendicular to the growth direction of the gel. The positional dependence of the order parameter of the collagen fibrils showed that the anisotropic collagen gel has an orientation gradient.     

The role of tumor necrosis factor-α for interleukin-10 production by murine dendritic cells

In the present study, we examined the role of tumor necrosis factor (TNF) in interleukin (IL)-10 production by dendritic cells (DCs) using bone-marrow derived DCs from wild type (WT) and TNF-α knockout (TNF-α^−/−) mice. Toll-like receptor (TLR) stimulation induced substantial level of IL-10 production by WT DCs, but significantly low level of IL-10 production by TNF-α^−/− DCs. In contrast, no significant difference was detected in IL-12 p40 production between WT and TNF-α^−/− DCs. Addition of TNF-α during TLR stimulation recovered the impaired ability of TNF-α^−/− DCs for IL-10 production. This recovery appeared to be associated with an activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/Akt following the TNF-α addition. Blocking these kinases significantly inhibited IL-10 production by TNF-α^−/− DCs stimulated with TLR ligands plus TNF-α. Thus, TNF-α may be a key molecule to regulate the balance between anti-inflammatory versus inflammatory cytokine production in DCs.     

Overexpression of an ADP-ribose pyrophosphatase, AtNUDX2, confers enhanced tolerance to oxidative stress in Arabidopsis plants

Mutant pqr-216 from an Arabidopsis activation-tagged line showed a phenotype of increased tolerance to oxidative stress after treatment with 3 μ m paraquat (PQ). Based on the phenotype of transgenic plants overexpressing the genes flanking the T-DNA insert, it was clear that enhanced expression of a Nudix (nucleoside diphosphates linked to some moiety X) hydrolase gene, AtNUDX2 (At5g47650), was responsible for the tolerance. It has been reported that the AtNUDX2 protein has pyrophosphatase activities towards both ADP-ribose and NADH ( Ogawa et al ., 2005 ). Interestingly, the pyrophosphatase activity toward ADP-ribose, but not NADH, was increased in pqr-216 and Pro _35S :AtNUDX2 plants compared with control plants. The amount of free ADP-ribose was lower in the Pro _35S :AtNUDX2 plants, while the level of NADH was similar to those in control plants under both normal conditions and oxidative stress. Depletion of NAD⁺ and ATP resulting from activation of poly(ADP-ribosyl)ation under oxidative stress was observed in the control Arabidopsis plants. Such alterations in the levels of these molecules were significantly suppressed in the Pro _35S :AtNUDX2 plants. The results indicate that overexpression of AtNUDX2 , encoding ADP-ribose pyrophosphatase, confers enhanced tolerance of oxidative stress on Arabidopsis plants, resulting from maintenance of NAD⁺ and ATP levels by nucleotide recycling from free ADP-ribose molecules under stress conditions.     

Comparison of cell lines for stable production of fucose-negative antibodies with enhanced ADCC

Several methods have been described to enhance antibody-dependent cellular cytotoxicity (ADCC) using different host cells that produce antibody with reduced levels of fucose on their carbohydrates. We compared the suitability of these methods for the serum-free fed-batch production of antibody for clinical trials and commercial uses. Recombinant anti-human CD20 chimeric IgG1-producing clones were established from host-cells that have been shown to produce more than 90% fucose-negative antibody. The cell lines were a FUT8 (alpha-1,6-fucosyltransferase) knockout Chinese hamster ovary (CHO) cell line, Ms704, and two Lens culinaris agglutinin (LCA)-resistant cell lines, one derived from a variant CHO line, Lec13 and the other from a rat hybridoma cell line, YB2/0. The amount of fucose-negative antibody produced by Lec13 and YB2/0 significantly decreased with the culture. The increase in fucosylation was due to remaining synthesis of GDP-fucose via de novo pathway for the CHO line and the elevation of FUT8 expression by the YB2/0 cells. In contrast, Ms704 cells stably produced fucose-negative antibody with a consistent carbohydrate structure until the end of the culture. The productivity of the Ms704 cells reached 1.76 g/L with a specific production rate (SPR) of 29 pg/cell/day for 17 days in serum-free fed-batch culture using a 1 L spinner bioreactor. Our results demonstrate that FUT8 knockout has the essential characteristics of host cells for robust manufacture of fucose-negative therapeutic antibodies with enhanced ADCC.     

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.