Suhn-Young Im

Glucocorticoids prevent NF‐κB activation by inhibiting the early release of platelet‐activating factor in response to lipopolysaccharide

Lipopolysaccharide (LPS) is a known inducer of numerous pro‐inflammatory events including the production of platelet‐activating factor (PAF). PAF released in response to LPS is a major contributor to the pathological events associated with endotoxemia. The present study demonstrates that dexmethasone (DEX) inhibited the LPS‐induced early plasma PAF raise in a dose‐ and time‐dependent manner. In addition, DEX prevented the subsequent PAF‐mediated pathological phenomena such as anaphylactic shock‐like symptoms, symptoms of disseminated intravascular coagulation and hemorrhage in renal medullae. DEX or the PAF antagonist BN 50739 significantly inhibited LPS‐induced NF‐κB activation. The inhibition of NF‐κB activation by DEX was overcome by the injection of exogenous PAF. Administration of PAF or LPS resulted in a rapid loss of IκBα protein. The LPS‐induced degradation of IκBα was prevented by pretreatment with BN 50739, suggesting that PAF is a critical intermediate in the LPS‐triggered degradation of IκBα protein. DEX prevented the LPS‐induced IκBα degradation, which was also reversed by exogenous PAF. Administration of DEX or BN 50739 caused an increase in cytoplasmic IκBα level. Our results indicate that DEX inhibits IκBα degradation and subsequent NF‐κB activation through blocking the initial release of PAF.

Essential role for platelet-activating factor-induced NF-κB activation in macrophage-derived angiogenesis

Activated monocyte‐macrophages have been implicated in tumor angiogenesis via their capacity to produce many potent angiogenic factors. However, the mechanisms leading to production of these angiogenic factors in macrophages remain to be elucidated. In this study, we demonstrated by use of a mouse Matrigel implantation model that mouse peritoneal macrophages induce angiogenesis. mRNA expression and protein synthesis of macrophage‐derived crucial angiogenic factors such as IL‐1, TNF‐α, basic fibroblast growth factor, and vascular endothelial growth factor (VEGF) were blocked by platelet‐activating factor (PAF) receptor antagonists. It was also observed that inhibitors of NF‐κB blocked macrophage production of these angiogenic factors. Gene expression and protein synthesis of the angiogenic factors cited above were also inhibited in IκBα‐mutated macrophages. VEGF is the most potent angiogenic factor in macrophage‐induced angiogenesis. PAF antagonists or NF‐κB inhibitors also inhibit the capacity of conditioned medium from LPS‐stimulated human peripheral blood monocytes to induce sprouting of porcine pulmonary arterial endothelial cells. Thesedata indicate that PAF‐induced NF‐κB activation is a common upstream pathway leading to the production of crucial macrophage‐derived angiogenic factors. This will provide an important clue for a better understanding of mechanisms involved in tumor angiogenesis.

Platelet-activating factor induces matrix metalloproteinase-9 expression through Ca2+- or PI3K-dependent signaling pathway in a human vascular endothelial cell line

Platelet‐activating factor (PAF) augments angiogenesis by promoting the synthesis of a variety of angiogenic factors, via the nuclear factor (NF)‐κB activation. Recently, we reported that PAF upregulates MMP‐9 expression in a NF‐κB‐dependent manner. In this study, we investigated the signaling pathway involved in PAF‐induced MMP‐9 expression in ECV304 cells. Our current data indicate that the Ca2+‐ or phosphatidylinositol 3‐kinase (PI3K)‐dependent signaling pathway is necessary for PAF‐induced MMP‐9 expression. Furthermore, PAF‐induced NF‐κB activation was blocked by selective inhibitors of Ca2+, PI3K, or extracellular signal‐regulated kinase (ERK). Our results suggest that PAF‐induced MMP‐9 expression, in a NF‐κB‐dependent manner, is regulated by Ca2+, PI3K and ERK signaling pathways.

Involvement of matrix metalloproteinase‐9 in platelet‐activating factor‐induced angiogenesis

Platelet‐activating factor (PAF) augments angiogenesis by promoting the synthesis of various angiogenic factors, via the activation of NF‐κB. In this study, we investigated the role of the matrix metalloproteinase (MMP)‐9, in PAF‐induced angiogenesis. PAF increased mRNA expression, protein synthesis, and MMP‐9 activity in ECV304 cells, in a NF‐κB‐dependent manner. PAF increased MMP‐9 promoter activity in ECV304, which was inhibited by WEB2107, and NF‐κB inhibitors. Transfected NF‐κB subunits, p65 or/and p50, increased luciferase activity in the reporter plasmid MMP‐9, resulting in an increase not only of MMP‐9 luciferase activity, but also of mRNA expression in MMP‐9. MMP‐9 or NF‐κB inhibitors significantly inhibited PAF‐induced angiogenesis, in a dose‐dependent manner, in an in vivo mouse Matrigel implantation model. In a parallel to the Matrigel implantation study, MMP‐9 or NF‐κB inhibitors inhibited PAF‐induced sprouting of porcine pulmonary arterial endothelial cells. These data indicate that NF‐κB‐dependent MMP‐9 plays a key role in PAF‐induced angiogenesis.

Cloning, expression and nucleotide sequences of two xylanase genes from Paenibacillus sp.

Two genes, xynA and xynB, encoding xylanases from Paenibacillus sp. KCTC 8848P were cloned and expressed in Escherichia coli, and their nucleotide sequences were determined. The xylanases of E. coli transformants were released into the extracellular culture fluid in the absence of xylan. The structural gene of xynA 636 bp, encoded a protein of 212 amino acids, while the xynB gene consisted of 951 bp open reading frame for a protein of 317 amino acids. The amino acid sequence of the xynAgene showed 83% similarity to the xylanase of Aeromonas caviae, and belonged to the family 11 glycosyl hydrolases. The deduced amino acid sequence of the xynB gene, however, showed 51% similarity to the xylanase of Rhodothermus marinus, and belonged to the family 10 glycosyl hydrolases.

Construction of an amylolytic industrial strain of Saccharomyces cerevisiae containing the Schwanniomyces occidentalis α-amylase gene

The gene encoding Schwanniomyces occidentalis α-amylase (AMY) was introduced into the chromosomal δ sequences of an industrial strain of Saccharomyces cerevisiae. To obtain a strain suitable for commercial use, an δ-integrative cassette devoid of bacterial DNA sequences was constructed that contains the AMY gene and aureobasidin A resistance gene (AUR1-C) as the selection marker. The AMY gene was expressed under the control of the alcohol dehydrogenase gene promoter (ADC1p). The α-amylase activity of Sacc. cerevisiae transformed with this integrative cassette was 6 times higher than that of Sch. occidentalis. The transformants (integrants) were mitotically stable after 100 generations in nonselective medium.

Platelet-activating factor-induced NF-κB activation enhances VEGF expression through a decrease in p53 activity

We investigated the role of p53 in nuclear factor (NF)‐κB dependent, platelet‐activating factor (PAF)‐induced vascular endothelial growth factor (VEGF) expression. Transfected NF‐κB subunits in ECV304 cells increased the tumor necrosis factor‐α promoter activity, which was completely inhibited by p53. Transfected p53 increased p53RE promoter activity, which was completely inhibited by NF‐κB subunits, indicating that cross‐regulation occurs between NF‐κB and p53. PAF‐induced increase in VEGF expression was correlated with decreased p53 activity. These data suggest that NF‐κB‐dependency of the PAF‐induced increase in VEGF expression is due to decreased p53 activity, which is reciprocally regulated by increased NF‐κB activity.

Protein Kinase CK2/PTEN Pathway Plays a Key Role in Platelet-Activating Factor-Mediated Murine Anaphylactic Shock

Platelet-activating factor (PAF) is a major mediator in the induction of fatal hypovolemic shock in murine anaphylaxis. This PAF-mediated effect has been reported to be associated with PI3K/Akt-dependent eNOS-derived NO. The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is phosphatidylinositol phosphate phosphatase, which negatively controls PI3K by dephosphorylating the signaling lipid, phosphatidylinositol 3,4,5-triphosphate. In this study, we examined the possible involvement of PTEN in PAF-mediated anaphylactic shock. Induction of anaphylaxis or PAF injection resulted in a rapid decrease in PTEN activity, followed by increases in PI3K activity and phosphorylation of Akt and eNOS. Systemic administration of adenoviruses carrying PTEN cDNA (adenoviral PTEN), but not the control AdLacZ, not only attenuated anaphylactic symptoms, but also reversed anaphylaxis- or PAF-induced changes in PTEN and PI3K activities, as well as phosphorylation of Akt and eNOS. We found that the decreased PTEN activity was associated with PTEN phosphorylation, the latter effect being prevented by the protein kinase CK2 inhibitor, DMAT. DMAT also inhibited anaphylactic symptoms as well as the anaphylaxis- or PAF-mediated PTEN/PI3K/Akt/eNOS signaling cascade. CK2 activity was increased by PAF. The present data provide, as the key mechanism underlying anaphylactic shock, PAF triggers the upstream pathway CK2/PTEN, which ultimately leads to the activation of PI3K/Akt/eNOS. Therefore, CK2/PTEN may be a potent target in the control of anaphylaxis and other many PAF-mediated pathologic conditions.

Construction of an industrial polyploid strain of Saccharomyces cerevisiae containing Saprolegnia ferax β-amylase gene and secreting β-amylase

An industrial polyploid strain of Saccharomyces cerevisiae containing Saprolegnia ferax β-amylase gene was developed by using two yeast integrating plasmids. One plasmid was constructed that contains the geneticin resistance gene (Gtr) as the selection marker and the ribosomal DNA (rDNA) portion that comprises the 18S rDNA as the recombination site. The other plasmid contains the aureobasidin A resistance gene (AUR1-C) as the selection marker and the chromosomal Ty δ sequence as the recombination site. The β-amylase activity of one clone of Saccharomyces cerevisiae transformed sequentially with these two plasmids was approx. 9 times higher than that of Saprolegnia ferax. This type of integration was mitotically stable even after 100 generations of cell multiplication under non-selective conditions.