Hyun-Mi Ko

Construction of a direct starch-fermenting industrial strain of Saccharomyces cerevisiae producing glucoamylase, α-amylase and debranching enzyme

To develop a strain of Saccharomyces cerevisiae that produces ethanol directly from starch, two integrative vectors were constructed to allow the simultaneous multiple integration of the Aspergillus awamori glucoamylase gene (GA1) and the Debaryomyces occidentalis α-amylase gene (AMY) and glucoamylase with debranching activity gene (GAM1) into the chromosomes of an industrial strain of S. cerevisiae. The GA1 and AMY genes were constitutively expressed under the ADC1 promoter in S. cerevisiae using the double δ-integration system. The GAM1 gene was constitutively expressed under the corresponding promoter using the double 18S rDNA-integration system. The recombinant industrial strain secreting biologically active α-amylase, glucoamylase and debranching enzyme was able to ferment starch to ethanol in a single step. The new strain produced 8% (v/v) ethanol (62.8xa0gxa0l−1) from 20% (w/v) soluble starch after 2xa0days, fermentation.

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.

Glutamine inhibits lipopolysaccharide-induced cytoplasmic phospholipase A2 activation and protects against endotoxin shock in mouse

ABSTRACT Glutamine (Gln) supplementation is known to play a beneficial role in a number of settings of critical illness as well as laboratory models of endotoxin shock. We have investigated a molecular mechanism of the protective role of Gln in lipopolysaccharide (LPS)-induced shock using a mouse model. To examine the effectiveness of Gln, Gln was administered before or after LPS injection. Treatment of Gln before, but not after, LPS injection resulted in inhibition of nuclear factor &kgr;B activation and tumor necrosis factor &agr; synthesis. In contrast, protection of animal from LPS-mediated death by Gln was observed when the Gln treatment was performed after LPS injection, suggesting that nuclear factor &kgr;B/tumor necrosis factor &agr; signaling does not play an important role in this process. LPS injection induced phosphorylation of cytoplasmic phospholipase A2 (cPLA2), which was blocked by Gln treatment after LPS injection. Similarly, the LPS-stimulated cPLA2 activity was also inhibited by Gln treatment after LPS injection. Moreover, a cPLA2 inhibitor not only inhibited LPS-induced activation of cPLA2, but also significantly prevented LPS-mediated death. These observations indicate that Gln has a capability to inhibit cPLA2 phosphorylation and activation and suggest that Gln might be of a great therapeutic value for controlling inflammatory diseases in which cPLA2 plays an important role in the pathogenesis of the diseases.ABBREVIATIONS-Gln, glutamine; cPLA2, cytoplasmic phospholipase A2; MAPK, mitogen-activated protein kinase; NF-&kgr;B, nuclear factor &kgr;B; TFMK, arachidonyltrifluoromethyl ketone; p65 AS, antisense against p65 components of NF-&kgr;B; p65 NS, p65 nonsense against p65 components of NF-&kgr;B; TNF-&agr;, tumor necrosis factor &agr;; LPS, lipopolysaccharide; PMSF, phenylmethylsulfonyl fluoride

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.

Efficient one-step starch utilization by industrial strains of Saccharomyces cerevisiae expressing the glucoamylase and α-amylase genes from Debaryomyces occidentalis

Amylolytic industrial polyploid strains of Saccharomyces cerevisiae (ATCC 4126, ATCC 9763 and ATCC 24858) expressing a glucoamylase gene (GAM1) or an α-amylase gene (AMY) from Debaryomyces occidentalis were developed. The glucoamylase activity of S.xa0cerevisiae ATCC 9763 expressing the GAM1 gene was 3.7-times higher than that of D.xa0occidentalis. On the other hand, α-amylase activity in the corresponding strain expressing the D.xa0occidentalisAMY gene increased 10-times relative to D.xa0occidentalis. These two recombinant yeast strains expressing the GAM1 gene and AMY gene, respectively were cultured simultaneously to produce both glucoamylase and α-amylase for efficient one-step utilization of starch. Growth, substrate utilization and enzyme activity of these strains are described.

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.

Epigallocatechin-3-gallate rescues LPS-impaired adult hippocampal neurogenesis through suppressing the TLR4-NF-κB signaling pathway in mice

Adult hippocampal dentate granule neurons are generated from neural stem cells (NSCs) in the mammalian brain, and the fate specification of adult NSCs is precisely controlled by the local niches and environment, such as the subventricular zone (SVZ), dentate gyrus (DG), and Toll-like receptors (TLRs). Epigallocatechin-3-gallate (EGCG) is the main polyphenolic flavonoid in green tea that has neuroprotective activities, but there is no clear understanding of the role of EGCG in adult neurogenesis in the DG after neuroinflammation. Here, we investigate the effect and the mechanism of EGCG on adult neurogenesis impaired by lipopolysaccharides (LPS). LPS-induced neuroinflammation inhibited adult neurogenesis by suppressing the proliferation and differentiation of neural stem cells in the DG, which was indicated by the decreased number of Bromodeoxyuridine (BrdU)-, Doublecortin (DCX)- and Neuronal Nuclei (NeuN)-positive cells. In addition, microglia were recruited with activatingTLR4-NF-κB signaling in the adult hippocampus by LPS injection. Treating LPS-injured mice with EGCG restored the proliferation and differentiation of NSCs in the DG, which were decreased by LPS, and EGCG treatment also ameliorated the apoptosis of NSCs. Moreover, pro-inflammatory cytokine production induced by LPS was attenuated by EGCG treatment through modulating the TLR4-NF-κB pathway. These results illustrate that EGCG has a beneficial effect on impaired adult neurogenesis caused by LPSinduced neuroinflammation, and it may be applicable as a therapeutic agent against neurodegenerative disorders caused by inflammation.

Parasympathectomy induces morphological changes and alters gene-expression profiles in the rat submandibular gland.

OBJECTIVEnThe chorda-lingual (CL) nerve carries parasympathetic fibers to the hilum of the sublingual and submandibular glands (SMGs) and evokes the secretion of saliva. The effect of cutting the CL nerve on the biological processes in SMGs was investigated by examining the gene-expression profiles in the SMGs after a surgical parasympathectomy.nnnMETHODSnAt day 3 after the CL nerve cut, the changes in the SMGs at both the experimental cut and contralateral control sides were analysed by microarray and light microscopy. The expression levels of 6 selected genes were confirmed by real-time PCR, Western blot and immunofluorescence staining.nnnRESULTSnThe wet weight of the parasympathectomised SMGs decreased significantly compared to that of the contralateral side (p<0.05). Histological analyses after the parasympathectomy showed a widened interacinar space as well as some atropic changes to the acini of the SMGs in the cut side. Microarray analysis revealed that twofold differential expression in mRNA expression in the parasympathectomized SMGs were detected in 88 genes (0.004%): 41 genes were overexpressed, 11 were underexpressed and 36 were unknown. Changes of the expression of 6 selected genes detected by Western blot and/or real-time PCR were consistent with the microarray data.nnnCONCLUSIONnThe important genes involved in biological processes for salivation were identified through a large-scale gene expression analysis.

Relaxin is up-regulated in the rat ovary by orthodontic tooth movement.

Relaxin (Rln) is an ovarian hormone that stimulates osteoclastic and osteoblastic activities and connective tissue turnover. To investigate the expression of Rln during orthodontic tooth movement, rats were implanted with orthodontic appliances that connected a spring from the upper incisors to the first molar with a 70 cN force. Rats in each group were killed 6, 48, and 144 h after activating the appliance, and the levels of Rln1 and Rln3 expression in the ovary were determined by real-time RT-PCR, northern blots, western blots, and immunofluorescence analyses. The amount of tooth movement induced by the orthodontic force increased in a time-dependent manner. The levels of Rln1 mRNA increased by 12-, 41-, and 263-fold at 6, 48, and 144 h, respectively, after orthodontic tooth movement. The time-dependent increase in the concentration of Rln 1 protein in the ovary was also confirmed by western blotting. Rln 1 was localized in the granulosa cells of the ovarian follicles, and the immunoreactivity against Rln 1 was increased by the movement. In contrast, the concentration of Rln 3 was below the level of detection. The results of this study suggest that local changes in periodontal tissues induced by orthodontic tooth movement may affect Rln1 expression in the ovary. However, further studies are needed to decipher the mechanisms involved and the possible contribution of the increased level of expression of Rln 1 to the tooth movement.