Hyong Kyu Kim

Helicobacter pylori CagA transfection of gastric epithelial cells induces interleukin‐8

To determine the effect of Helicobacter pylori CagA expression on interleukin‐8 (IL‐8) induction in AGS cells, cagA and five of its fragments from strains 147A and 147C that vary in the 3′ repeat region were cloned into the eukaryotic expression plasmid pSP65SRα. IL‐8, but not RANTES or IL‐Iβ, levels were increased in AGS cells transfected with 147A‐cagA and to a greater extent with 147C‐cagA, compared with negative controls. The 5′ b fragment from the two strains had similar effects, but the 3′ d and e fragments from 147C CagA had greater effects than those from 147A‐CagA. When the Western CagA‐specific sequence (WSS) of 147C‐cagA was replaced with East Asian CagA‐specific sequence (ESS) and cloned into pSP65SRα as an East/West chimera, there was no significant effect on IL‐8 production. Use of specific inhibitors indicates that Src kinase activation, and the mitogen‐activated protein (MAP) kinase and NF‐κB pathways are the major intermediates for CagA effects on IL‐8 induction, but the p38 MAP kinase pathway has little effect. These results indicate a direct CagA effect on IL‐8 induction by gastric epithelial cells, and indicate signal pathway loci that can be targeted for amelioration.

Monophyly of clade III nematodes is not supported by phylogenetic analysis of complete mitochondrial genome sequences

BackgroundThe orders Ascaridida, Oxyurida, and Spirurida represent major components of zooparasitic nematode diversity, including many species of veterinary and medical importance. Phylum-wide nematode phylogenetic hypotheses have mainly been based on nuclear rDNA sequences, but more recently complete mitochondrial (mtDNA) gene sequences have provided another source of molecular information to evaluate relationships. Although there is much agreement between nuclear rDNA and mtDNA phylogenies, relationships among certain major clades are different. In this study we report that mtDNA sequences do not support the monophyly of Ascaridida, Oxyurida and Spirurida (clade III) in contrast to results for nuclear rDNA. Results from mtDNA genomes show promise as an additional independently evolving genome for developing phylogenetic hypotheses for nematodes, although substantially increased taxon sampling is needed for enhanced comparative value with nuclear rDNA. Ultimately, topological incongruence (and congruence) between nuclear rDNA and mtDNA phylogenetic hypotheses will need to be tested relative to additional independent loci that provide appropriate levels of resolution.ResultsFor this comparative phylogenetic study, we determined the complete mitochondrial genome sequences of three nematode species, Cucullanus robustus (13,972 bp) representing Ascaridida, Wellcomiasiamensis (14,128 bp) representing Oxyurida, and Heliconema longissimum (13,610 bp) representing Spirurida. These new sequences were used along with 33 published nematode mitochondrial genomes to investigate phylogenetic relationships among chromadorean orders. Phylogenetic analyses of both nucleotide and amino acid sequence datasets support the hypothesis that Ascaridida is nested within Rhabditida. The position of Oxyurida within Chromadorea varies among analyses; in most analyses this order is sister to the Ascaridida plus Rhabditida clade, with representative Spirurida forming a distinct clade, however, in one case Oxyurida is sister to Spirurida. Ascaridida, Oxyurida, and Spirurida (the sampled clade III taxa) do not form a monophyletic group based on complete mitochondrial DNA sequences. Tree topology tests revealed that constraining clade III taxa to be monophyletic, given the mtDNA datasets analyzed, was a significantly worse result.ConclusionThe phylogenetic hypotheses from comparative analysis of the complete mitochondrial genome data (analysis of nucleotide and amino acid datasets, and nucleotide data excluding 3rd positions) indicates that nematodes representing Ascaridida, Oxyurida and Spirurida do not share an exclusive most recent common ancestor, in contrast to published results based on nuclear ribosomal DNA. Overall, mtDNA genome data provides reliable support for nematode relationships that often corroborates findings based on nuclear rDNA. It is anticipated that additional taxonomic sampling will provide a wealth of information on mitochondrial genome evolution and sequence data for developing phylogenetic hypotheses for the phylum Nematoda.

Mechanism and treatment for learning and memory deficits in mouse models of Noonan syndrome

In Noonan syndrome (NS) 30–50% of subjects show cognitive deficits of unknown etiology and with no known treatment. Here, we report that knock-in mice expressing either of two NS-associated mutations in Ptpn11, which encodes the nonreceptor protein tyrosine phosphatase Shp2, show hippocampal-dependent impairments in spatial learning and deficits in hippocampal long-term potentiation (LTP). In addition, viral overexpression of an NS-associated allele PTPN11D61G in adult mouse hippocampus results in increased baseline excitatory synaptic function and deficits in LTP and spatial learning, which can be reversed by a mitogen-activated protein kinase kinase (MEK) inhibitor. Furthermore, brief treatment with lovastatin reduces activation of the GTPase Ras–extracellular signal-related kinase (Erk) pathway in the brain and normalizes deficits in LTP and learning in adult Ptpn11D61G/+ mice. Our results demonstrate that increased basal Erk activity and corresponding baseline increases in excitatory synaptic function are responsible for the LTP impairments and, consequently, the learning deficits in mouse models of NS. These data also suggest that lovastatin or MEK inhibitors may be useful for treating the cognitive deficits in NS.

Autophagy regulates amyotrophic lateral sclerosis-linked fused in sarcoma-positive stress granules in neurons.

Mutations in fused in sarcoma (FUS), a DNA/RNA binding protein, have been associated with familial amyotrophic lateral sclerosis (fALS), which is a fatal neurodegenerative disease that causes progressive muscular weakness and has overlapping clinical and pathologic characteristics with frontotemporal lobar degeneration. However, the role of autophagy in regulation of FUS-positive stress granules (SGs) and aggregates remains unclear. We found that the ALS-linked FUS(R521C) mutation causes accumulation of FUS-positive SGs under oxidative stress, leading to a disruption in the release of FUS from SGs in cultured neurons. Autophagy controls the quality of proteins or organelles; therefore, we checked whether autophagy regulates FUS(R521C)-positive SGs. Interestingly, FUS(R521C)-positive SGs were colocalized to RFP-LC3-positive autophagosomes. Furthermore, FUS-positive SGs accumulated in atg5(-/-) mouse embryonic fibroblasts (MEFs) and in autophagy-deficient neurons. However, FUS(R521C) expression did not significantly impair autophagic degradation. Moreover, autophagy activation with rapamycin reduced the accumulation of FUS-positive SGs in an autophagy-dependent manner. Rapamycin further reduced neurite fragmentation and cell death in neurons expressing mutant FUS under oxidative stress. Overall, we provide a novel pathogenic mechanism of ALS associated with a FUS mutation under oxidative stress, as well as therapeutic insight regarding FUS pathology associated with excessive SGs.

Isolation and Characterization of Novel H3N1 Swine Influenza Viruses from Pigs with Respiratory Diseases in Korea

ABSTRACT Pigs can play an important role in the genetic reassortment of influenza viruses and as a reservoir for another lineage of influenza viruses that have the ability to reassort and be transmitted between species. In March and April 2006, novel H3N1 influenza A viruses were isolated from pigs with respiratory diseases at two different commercial swine farms in Korea. Genetic and phylogenetic analyses of the sequences of all eight viral RNA segments showed that the novel H3N1 swine influenza viruses were reassortants that acquired the hemagglutinin gene from an H3 human-like virus and other genes from swine influenza viruses that are currently circulating in Korea. Serologic and virologic tests in the infected farms suggested that pig-to-pig and farm-to-farm transmissions occurred. Clinical signs in pigs and experimentally infected mice suggest the potential to transmit the virus between swine and other mammalian hosts. To our knowledge, this is the first report of the isolation of the swine H3N1 subtype from domestic pigs under field conditions in Korea. Further surveillance will be needed to determine whether this novel subtype will continue to circulate in the swine population.

The transport of Staufen2‐containing ribonucleoprotein complexes involves kinesin motor protein and is modulated by mitogen‐activated protein kinase pathway

There is increasing evidence showing that mRNA is transported to the neuronal dendrites in ribonucleoprotein (RNP) complexes or RNA granules, which are aggregates of mRNA, rRNA, ribosomal proteins, and RNA‐binding proteins. In these RNP complexes, Staufen, a double‐stranded RNA‐binding protein, is believed to be a core component that plays a key role in the dendritic mRNA transport. This study investigated the molecular mechanisms of the dendritic mRNA transport using green fluorescent protein‐tagged Staufen2 produced employing a Sindbis viral expression system. The kinesin heavy chain was found to be associated with Staufen2. The inhibition of kinesin resulted in a significant decrease in the level of dendritic transport of the Staufen2‐containing RNP complexes in neurons under non‐stimulating or stimulating conditions. This suggests that the dendritic transport of the Staufen2‐containing RNP complexes use kinesin as a motor protein. A mitogen‐activated protein kinase inhibitor, PD98059, inhibited the activity‐induced increase in the amount of both the Staufen2‐containing RNP complexes and Ca2+/calmodulin‐dependent protein kinase II α‐subunit mRNA in the distal dendrites of cultured hippocampal neurons. Overall, these results suggest that dendritic mRNA transport is mediated via the Staufen2 and kinesin motor proteins and might be modulated by the neuronal activity and mitogen‐activated protein kinase pathway.

Phosphorylation of RhoGDI1 by p21-activated kinase 2 mediates basic fibroblast growth factor-stimulated neurite outgrowth in PC12 cells.

We previously showed that p21-activated kinase 2 (PAK2), a major PAK isoform expressed in PC12 cells, mediates neurite outgrowth via Rac1 GTPase. RhoGDI1 forms a complex with Rac1, resulting in its inhibition. Rac1 activation requires dissociation from RhoGDI1. Here, we show that PAK2 mediates basic fibroblast growth factor (bFGF)-stimulated neurite outgrowth via phosphorylation of RhoGDI1. RhoGDI1 was shown to be associated with PAK2, with phosphorylation of Ser34 and Ser101 by active PAK2 evident in vitro and in vivo. A RhoGDI1 phosphomimetic mutant (S34E/S101E) was dissociated from Rac1/Cdc42, whereas the wild-type or a nonphosphorylatable mutant (S34A/S101A) formed a tight complex. Consistent with this, PC12 cells expressing the phosphomimetic mutant displayed Rac1/Cdc42 activation in response to bFGF stimulation. Neurite outgrowth was also enhanced in PC12 cells expressing the phosphomimetic mutant. These results suggest that PAK2-mediated RhoGDI1 phosphorylation stimulates dissociation of RhoGDI1-Rac1/Cdc42 complex accompanied by relief of inhibitory effect on Rac1/Cdc42, which promotes neuronal differentiation.

Transcriptome analysis and identification of regulators for long-term plasticity in Aplysia kurodai

The marine mollusk Aplysia is a useful model organism for studying the cellular bases of behavior and plasticity. However, molecular studies of Aplysia have been limited by the lack of genomic information. Recently, a large scale characterization of neuronal transcripts was performed in A. californica. Here, we report the analysis of a parallel set of neuronal transcripts from a closely related species A. kurodai found in the northwestern Pacific. We collected 4,859 nonredundant sequences from the nervous system tissue of A. kurodai. By performing microarray and real-time PCR analyses, we found that ApC/EBP, matrilin, antistasin, and eIF3e clones were significantly up-regulated and a BAT1 homologous clone was significantly down-regulated by 5-HT treatment. Among these, we further demonstrated that the Ap-eIF3e plays a key role in 5-HT-induced long-term facilitation (LTF) as a positive regulator.

Role of Staufen in dendritic mRNA transport and its modulation.

Staufen is a double-stranded RNA-binding protein and a core component in various RNP complexes or RNA granules, and plays an important role in dendritic mRNA transport. In this study, a ribosomal marker and a dominant-negative form of Staufen (stau-RBD), containing the RNA-binding domains, but lacking a microtubule-association domain, was used to determine the role of Staufen in dendritic mRNA transport. The results showed that the overexpression of stau-RBD significantly decreased the levels of ribosomal staining in the dendrites, which was illustrated by Y10B immunostaining. In contrast, the overexpression of Staufen increased the ribosomal level. The regulatory mechanisms of the dendritic mRNA transport were examined using a GFP-tagged Staufen (GFP-Stau), which was produced by means of a Sindbis viral expression system. Depolarization increased the amount of Staufen-containing the RNP complexes and endogenous Staufen in the dendrites. This increase was independent of protein synthesis. This suggests that dendritic mRNA transport is mediated via Staufen, and is regulated by the neuronal activity.

Phosphorylation of β-catenin at serine 663 regulates its transcriptional activity.

β-Catenin, a component of Wnt signaling, plays a key role in colorectal carcinogenesis. The phosphorylation status of β-catenin determines its fate and affects its cellular function, and serine 675 (S675) was previously identified as a common target of p21-activated kinase 1 (PAK1) and protein kinase A. In the present study, we explored the PAK1-specific phosphorylation site(s) in β-catenin. Active PAK1 T423E but not inactive PAK1 K299R interacted with and phosphorylated β-catenin. Mutagenesis followed by a kinase assay revealed that PAK1 phosphorylated S663 in addition to S675, and an anti-phospho-β-catenin(S663) antibody detected the phosphorylation of S663 downstream of PAK1 in various human colon cancer cells. Furthermore, the Wnt3a-stimulated S663 phosphorylation was inhibited by the PAK1-specific inhibitor, IPA-3, but not by H-89 or LY294002. The non-phosphorylatable mutant forms of β-catenin, S663A, S675A and S663/675A, showed similar defects in their PAK1-induced TCF/LEF transactivation, whereas the phosphomimetic form of β-catenin, S663D, demonstrated a transcriptional activity that was comparable to that of β-catenin S675D and β-catenin S663D/S675D. Taken together, these results provide evidence that PAK1 specifically phosphorylates β-catenin at S663 and that this phosphorylation is essential for the PAK1-mediated transcriptional activation of β-catenin.