Oxidative stress augments toll-like receptor 8 mediated neutrophilic responses in healthy subjects

Background

Excessive oxidative stress has been reported to be generated in inflamed tissues and contribute to the pathogenesis of inflammatory lung diseases, exacerbations of which induced by viral infections are associated with toll-like receptor (TLR) activation. Among these receptors, TLR8 has been reported as a key receptor that recognizes single-strand RNA virus. However, it remains unknown whether TLR8 signaling is potentiated by oxidative stress. The aim of this study is to examine whether oxidative stress modulates TLR8 signaling in vitro.

Methods

Human peripheral blood neutrophils were obtained from healthy non-smokers and stimulated with TLR 7/8 agonist imidazoquinoline resiquimod (R848) in the presence or absence of hydrogen peroxide (H₂O₂). Neutrophilic responses including cytokine release, superoxide production and chemotaxis were examined, and the signal transduction was also analyzed.

Results

Activation of TLR8, but not TLR7, augmented IL-8 release. The R848-augmented IL-8 release was significantly potentiated by pretreatment with H₂O₂ (p < 0.01), and N-acetyl-L-cysteine reversed this potentiation. The combination of H₂O₂ and R848 significantly potentiated NF-kB phosphorylation and IkBα degradation. The H₂O₂-potentiated IL-8 release was suppressed by MG-132, a proteosome inhibitor, and by dexamethasone. The expressions of TLR8, myeloid differentiation primary response gene 88 (MyD88), and tumor necrosis factor receptor-associated factor 6 (TRAF6) were not affected by H₂O₂.

Conclusion

TLR8-mediated neutrophilic responses were markedly potentiated by oxidative stress, and the potentiation was mediated by enhanced NF-kB activation. These results suggest that oxidative stress might potentiate the neutrophilic inflammation during viral infection.     

Annual and spatial variation in shoot demography associated with masting in Betula grossa: comparison between mature trees and saplings

Backgrounds and Aims

Shoot demography affects the growth of the tree crown and the number of leaves on a tree. Masting may cause inter-annual and spatial variation in shoot demography of mature trees, which may in turn affect the resource budget of the tree. The aim of this study was to evaluate the effect of masting on the temporal and spatial variations in shoot demography of mature Betula grossa.

Methods

The shoot demography was analysed in the upper and lower parts of the tree crown in mature trees and saplings over 7 years. Mature trees and saplings were compared to differentiate the effect of masting from the effect of exogenous environment on shoot demography. The fate of different shoot types (reproductive, vegetative, short, long), shoot length and leaf area were investigated by monitoring and by retrospective survey using morphological markers on branches. The effects of year and branch position on demographic parameters were evaluated.

Key Results

Shoot increase rate, production of long shoots, bud mortality, length of long shoots and leaf area of a branch fluctuated periodically from year to year in mature trees over 7 years, in which two masting events occurred. Branches within a crown showed synchronized annual variation, and the extent of fluctuation was larger in the upper branches than the lower branches. Vegetative shoots varied in their bud differentiation each year and contributed to the dynamic shoot demography as much as did reproductive shoots, suggesting physiological integration in shoot demography through hormonal regulation and resource allocation.

Conclusions

Masting caused periodic annual variation in shoot demography of the mature trees and the effect was spatially variable within a tree crown. Since masting is a common phenomenon among tree species, annual variation in shoot demography and leaf area should be incorporated into resource allocation models of mature masting trees.     

Characterization of a novel acid phosphatase from embryonic axes of kidney bean exhibiting vanadate-dependent chloroperoxidase activity

A novel colorless acid phosphatase (KeACP), which was distinct from the kidney bean purple acid phosphatase, was purified to apparent homogeneity and cloned from embryonic axes of kidney bean (Phaseolus vulgaris L. Ohfuku) during germination. When orthovanadate (VO(4)(-3)) is added to the apo form of the enzyme, KeACP uniquely exhibits the chloroperoxidase activity with loss of phosphatase activity. This is the first demonstration that KeACP is a vanadate-dependent chloroperoxidase in plants to be characterized and suggests that KeACP may play a role in modifying a wide variety of chlorinated compounds that are present in higher plants. The enzyme is a dimer that presents three forms made up of the combination of the dominant 56-kDa and the minor 45-kDa subunits, and both subunits contain carbohydrate. The full-length cDNA of the KeACP gene is 1641 nucleotides, and this sequence is predicted to encode a protein having 457 amino acid residues (52,865 Da), including a signal peptide. The complete nucleotide sequence of the genomic DNA (3228 bp) of KeACP consists of seven exons and six introns. Northern blot analysis demonstrated that the KeACP gene was expressed specifically in embryonic axes of the kidney bean, and its expression coincided with elongation of the embryonic axis during germination.     

Immunolocalization of CXC chemokine and recruitment of polymorphonuclear leukocytes in the rat molar periodontal tissue after topical application of lipopolysaccharide

This study investigated the recruitment of polymorphonuclear leukocytes (PMNs) and the immunolocalization of CXC chemokines, including macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant-2 (CINC-2) in rat periodontal tissue after topical application of lipopolysaccharide (LPS; 5 mg/ml) from Escherichia coli into the rat molar gingival sulcus. In normal periodontal tissues, a small number of MIP-2- and CINC-2-positive cells were seen in junctional epithelium (JE), especially in its coronal half. After topical application of LPS, a prominent increase of MIP-2- and CINC-2-positive JE cells was observed. Almost all JE cells strongly expressed them at day 1 and day 2, and then the number of chemokine-positive cells returned to normal at day 7. Corresponding to these chemokine expressions, LPS application induced a significant increase in the number of PMNs in the sub-JE area from 1 h to 2 days and a significant increase in JE area from 3 h to 5 days, indicating a dynamic flow of PMNs from the sub-JE area into JE. These findings indicated that JE cells produced MIP-2 and CINC-2 in response to LPS stimulation and suggested that MIP-2 and CINC-2 may be responsible for PMN migration toward the periodontal pathogen and may play an important role in the initiation of inflammation and subsequent periodontal tissue destruction.     

Nucleolin and the packaging signal, psi, promote the budding of human immunodeficiency virus type-1 (HIV-1)

Gag proteins of human immunodeficiency virus type 1 (HIV-1) play a pivotal role in the budding of the virion, in which the zinc finger motifs of the gag proteins recognize the packaging signal of genomic RNA. Nucleolin, an RNA-binding protein, is identified as a cellular protein that binds to murine leukemia virus (MuLV) gag proteins and regulates the viral budding, suggesting that HIV-1 gag proteins, the packaging signal, psi and nucleolin affect the budding of HIV-1. Here we report that nucleolin enhances the release of HIV-1 virions which contain psi. Furthermore, nucleolin and gag proteins form a complex incorporated into virions, and nucleolin promotes the infectivity of HIV-1. Our results suggest that an empty particle which contains neither nucleolin nor the genomic RNA is eliminated during the budding process, and this mechanism is beneficial for escape from the host immune response against HIV-1.     

L-Glutamate in the extracellular space regulates endogenous D-aspartate homeostasis in rat pheochromocytoma MPT1 cells

In previous studies [FEBS Lett. 434 (1998) 231, Arch. Biochem. Biophys. 404 (2002) 92], we demonstrated for the first time that D-aspartate (D-Asp) is synthesized in cultured mammalian cell lines, such as pheochromocytoma 12 (PC12) and its subclone, MPT1. Our current focus is analysis of the dynamics of D-Asp homeostasis in these cells. In this communication, we show that L-glutamate (Glu) and L-Glu transporter substrates in the extracellular space regulate the homeostasis of endogenous D-Asp in MPT1 cells. D-Asp is apparently in dynamic homeostasis, whereby endogenous D-Asp is constantly released into the extracellular space by an undefined mechanism, and continuously and intensively taken up into cells by an L-Glu transporter. Under these conditions, L-Glu and its transporter substrates in the medium may competitively inhibit the uptake of D-Asp via the transporter, resulting in accumulation of the amino acid in the extracellular space. We additionally demonstrate that DL-TBOA, a well-established L-Glu transporter inhibitor, is taken up by the transporter during long time intervals, but not on a short time-scale.     

Synthesis and biological activity of a novel squalene epoxidase inhibitor, FR194738

The synthesis and biological properties of a novel squalene epoxidase inhibitor, FR194738, are described. This compound displayed potent in vitro inhibitory activities against squalene epoxidase and cholesterol synthesis, and lowered plasma cholesterol and triglyceride levels in dogs.     

A genetic link between Tbx1 and fibroblast growth factor signaling

Tbx1 haploinsufficiency causes aortic arch abnormalities in mice because of early growth and remodeling defects of the fourth pharyngeal arch arteries. The function of Tbx1 in the development of these arteries is probably cell non-autonomous, as the gene is not expressed in structural components of the artery but in the surrounding pharyngeal endoderm. We hypothesized that Tbx1 may trigger signals from the pharyngeal endoderm directed to the underlying mesenchyme. We show that the expression patterns of Fgf8 and Fgf10, which partially overlap with Tbx1 expression pattern, are altered in Tbx1(-/-) mutants. In particular, Fgf8 expression is abolished in the pharyngeal endoderm. To understand the significance of this finding for the pathogenesis of the mutant Tbx1 phenotype, we crossed Tbx1 and Fgf8 mutants. Double heterozygous Tbx1(+/-);Fgf8(+/-) mutants present with a significantly higher penetrance of aortic arch artery defects than do Tbx1(+/-);Fgf8(+/+) mutants, while Tbx1(+/+);Fgf8(+/-) animals are normal. We found that Fgf8 mutation increases the severity of the primary defect caused by Tbx1 haploinsufficiency, i.e. early hypoplasia of the fourth pharyngeal arch arteries, consistent with the time and location of the shared expression domain of the two genes. Hence, Tbx1 and Fgf8 interact genetically in the development of the aortic arch. Our data provide the first evidence of a genetic link between Tbx1 and FGF signaling, and the first example of a modifier of the Tbx1 haploinsufficiency phenotype. We speculate that the FGF8 locus might affect the penetrance of cardiovascular defects in individuals with chromosome 22q11 deletions involving TBX1.     

Cell density inversely regulates D- and L-aspartate levels in rat pheochromocytoma MPT1 cells

In a previous report (FEBS Lett. 434 (1998) 231), we demonstrated for the first time that D-aspartate (D-Asp) is synthesized in rat pheochromocytoma 12 (PC12) cells. This unique amino acid is believed to act as a novel messenger in mammalian cell regulation. However, the dynamics of D-Asp homeostasis in mammalian cells is yet to be elucidated. In this communication, we demonstrate that D-Asp is also synthesized in MPT1 cells (a subclone of PC12 cells) and that the D- and L-Asp levels in cells are regulated by cell density of the culture. Our data show that D-Asp levels increase, while in contrast, L-Asp levels decrease as a function of increased cell density. Conversely, in PC12 cells, which do not express the glutamate transporter involved in the incorporation of D- and L-Asp into cells, L-Asp levels decrease upon cell density increase while D-Asp concentrations remain almost unchanged. The results indicate that the biochemical behaviors of D- and L-Asp in mammalian cells are distinct and that the cellular levels of these stereoisomers appear to be under different control mechanisms.