Proteins interact with each other within a cell, and those interactions give rise to the biological function and dynamical behavior of cellular systems. Generally, the protein interactions are temporal, spatial, or condition dependent in a specific cell, where only a small part of interactions usually take place under certain conditions. Recently, although a large amount of protein interaction data have been collected by high-throughput technologies, the interactions are recorded or summarized under various or different conditions and therefore cannot be directly used to identify signaling pathways or active networks, which are believed to work in specific cells under specific conditions. However, protein interactions activated under specific conditions may give hints to the biological process underlying corresponding phenotypes. In particular, responsive functional modules consist of protein interactions activated under specific conditions can provide insight into the mechanism underlying biological systems, e.g. protein interaction subnetworks found for certain diseases rather than normal conditions may help to discover potential biomarkers. From computational viewpoint, identifying responsive functional modules can be formulated as an optimization problem. Therefore, efficient computational methods for extracting responsive functional modules are strongly demanded due to the NP-hard nature of such a combinatorial problem. In this review, we first report recent advances in development of computational methods for extracting responsive functional modules or active pathways from protein interaction network and microarray data. Then from computational aspect, we discuss remaining obstacles and perspectives for this attractive and challenging topic in the area of systems biology.

"Two-cysteine" peroxiredoxins are antioxidant enzymes that exert a cytoprotective effect in many models of oxidative stress. However, under highly oxidizing conditions they can be inactivated through hyperoxidation of their peroxidatic active site cysteine residue. Sulfiredoxin can reverse this hyperoxidation, thus reactivating peroxiredoxins. Here we review recent investigations that have shed further light on sulfiredoxin's role and regulation. Studies have revealed sulfiredoxin to be a dynamically regulated gene whose transcription is induced by a variety of signals and stimuli. Sulfiredoxin expression is regulated by the transcription factor AP-1, which mediates its up-regulation by synaptic activity in neurons, resulting in protection against oxidative stress. Furthermore, sulfiredoxin has been identified as a new member of the family of genes regulated by Nuclear factor erythroid 2-related factor (Nrf2) via a conserved cis-acting antioxidant response element (ARE). As such, sulfiredoxin is likely to contribute to the net antioxidative effect of small molecule activators of Nrf2. As discussed here, the proximal AP-1 site of the sulfiredoxin promoter is embedded within the ARE, as is common with Nrf2 target genes. Other recent studies have shown that sulfiredoxin induction via Nrf2 may form an important part of the protective response to oxidative stress in the lung, preventing peroxiredoxin hyperoxidation and, in certain cases, subsequent degradation. We illustrate here that sulfiredoxin can be rapidly induced in vivo by administration of CDDO-TFEA, a synthetic triterpenoid inducer of endogenous Nrf2, which may offer a way of reversing peroxiredoxin hyperoxidation in vivo following chronic or acute oxidative stress.

Curcumin, a natural compound extracted from rhizomes of curcuma Curcuma species, has been shown to possess potent anti-inflammatory, anti-tumor and anti-oxidative properties. However, the mechanism of action of the compound remains poorly understood. In this report, we have analyzed the effects of curcumin on the cell proliferation of Burkitt's lymphoma Raji cells. The results demonstrated that curcumin could effectively inhibit the growth of Raji cells in a dose- and time-dependent manner. Further studies indicated that curcumin treatment resulted in apoptosis of cells. Biochemical analysis showed that the expression of Bax, Bid and cytochrome C were up-regulated, while the expression of oncogene c-Myc was down regulated after curcumin treatment. Furthermore, poly (ADP-ribose) polymerase (PARP) cleavage was induced by the compound. Interestingly, the antiapoptotic Bcl-2 expression was not significantly changed in Raji cells after curcumin treatment. These results suggested that the mechanism of action of curcumin was to induce mitochondrial damage and therefore led to Raji cell apoptosis. We further investigated the in vivo effects of curcumin on the growth of xenograft tumors in nude mice. The results showed that curcumin could effectively inhibit tumor growth in the xenograft mouse model. The overall results showed that curcumin could suppress the growth of Burkitt's lymphoma cells in both in vitro and in vivo systems.

Apolipoprotein (apo) C-III is a marker protein of triacylglycerol (TG)-rich lipoproteins and high-density lipoproteins (HDL), and has been proposed as a risk factor of coronary heart disease. To compare the physiologic role of reconstituted HDL (rHDL) with or without apoC-III, we synthesized rHDL with molar ratios of apoA-I:apoC-III of 1:0, 1:0.5, 1:1, and 1:2. Increasing the apoC-III content in rHDL produced smaller rHDL particles with a lower number of apoA-I molecules. Furthermore, increasing the molar ratio of apoC-III in rHDL enhanced the surfactant-like properties and the ability to lyse dimyristoyl phosphatidylcholine. Furthermore, rHDL containing apoC-III was found to be more resistant to particle rearrangement in the presence of low-density lipoprotein (LDL) than rHDL that contained apoA-I alone. In addition, the lecithin:cholesterol acyltransferase (LCAT) activation ability was reduced as the apoC-III content of the rHDL increased; however, the CE transfer ability was not decreased by the increase of apoC-III. Finally, rHDL containing apoC-III aggravated the production of MDA in cell culture media, which led to increased cellular uptake of LDL. Thus, the addition of apoC-III to rHDL induced changes in the structural and functional properties of the rHDL, especially in particle size and rearrangement and LCAT activation. These alterations may lead to beneficial functions of HDL, which is involved in anti-atherogenic properties in the circulation.

Over-expression of phospholipase D (PLD) 1 or PLD2 down-regulated CKII activity in NIH3T3 cells. The same results were found with catalytically inactive mutants of PLD isozymes, indicating that the catalytic activity of PLD is not required for PLD-mediated CKII inhibition. Consistent with this, 1-butanol did not alter CKII activity. The reduction in CKII activity in PLD-over-expressing NIH3T3 cells was due to reduced protein level, but not mRNA level, of the $CKII{\beta}$ subunit. This PLD-induced $CKII{\beta}$ degradation was mediated by ubiquitin-proteasome machinery, but MAP kinase and mTOR were not involved in $CKII{\beta}$ degradation. PLD isozymes interacted with the $CKII{\beta}$ subunit. Immunocytochemical staining revealed that PLD and $CKII{\beta}$ colocalize in the cytoplasm of NIH3T3 cells, especially in the perinuclear region. PLD binding to $CKII{\beta}$ inhibited $CKII{\beta}$ autophosphorylation, which is known to be important for $CKII{\beta}$ stability. In summary, the current data indicate that PLD isozymes can down-regulate CKII activity through the acceleration of $CKII{\beta}$ degradation by ubiquitin-proteasome machinery.

The interstitial cells of Cajal (ICC) are pacemaking cells required for gastrointestinal motility. The possibility of whether DA-9701, a novel prokinetic agent formulated with Pharbitis Semen and Corydalis Tuber, modulates pacemaker activities in the ICC was tested using the whole cell patch clamp technique. DA-9701 produced membrane depolarization and increased tonic inward pacemaker currents in the voltage-clamp mode. The application of flufenamic acid, a non-selective cation channel blocker, but not niflumic acid, abolished the generation of pacemaker currents induced by DA-9701. Pretreatment with a $Ca^{2+}$-free solution and thapsigargin, a $Ca^{2+}$-ATPase inhibitor in the endoplasmic reticulum, abolished the generation of pacemaker currents. In addition, the tonic inward currents were inhibited by U-73122, an active phospholipase C inhibitor, but not by $GDP-{\beta}-S$, which permanently binds G-binding proteins. Furthermore, the protein kinase C inhibitors, chelerythrine and calphostin C, did not block the DA-9701-induced pacemaker currents. These results suggest that DA-9701 might affect gastrointestinal motility by the modulation of pacemaker activity in the ICC, and the activation is associated with the non-selective cationic channels via external $Ca^{2+}$ influx, phospholipase C activation, and $Ca^{2+}$ release from internal storage in a G protein-independent and protein kinase C-independent manner.

The dual-vector system-II (DVS-II), which allows efficient display of Fab antibodies on phage, has been reported previously, but its practical applicability in a phage-displayed antibody library has not been verified. To resolve this issue, we created two small combinatorial human Fab antibody libraries using the DVS-II, and isolation of target-specific antibodies was attempted. Biopanning of one antibody library, termed DVFAB-1L library, which has a $1.3{\times}10^7$ combinatorial antibody complexity, against fluorescein-BSA resulted in successful isolation of human Fab clones specific for the antigen despite the presence of only a single light chain in the library. By using the unique feature of the DVS-II, an antibody library of a larger size, named DVFAB-131L, which has a $1.5{\times}10^9$ combinatorial antibody complexity, was also generated in a rapid manner by combining $1.3{\times}10^7$ heavy chains and 131 light chains and more diverse anti-fluorescein-BSA Fab antibody clones were successfully obtained. Our results demonstrate that the DVS-II can be applied readily in creating phage-displayed antibody libraries with much less effort, and target-specific antibody clones can be isolated reliably via light chain promiscuity of antibody molecules.

Voltage-dependent anion channels (VDACs) are reported to be porin-type, ${\beta}$-barrel diffusion pores. They are prominently localized in the outer mitochondrial membrane and are involved in metabolite exchange between the organelle and the cytosol. In this study, we have investigated a family of VDAC isoforms in Arabidopsis thaliana (AtVDAC). We have shown that the heterologous expression of AtVDAC proteins can functionally complement a yeast mutant lacking the endogenous mitochondrial VDAC gene. AtVDACs tagged with GFP were localized to mitochondria in both yeast and plant cells. We also looked at the response of AtVDACs to biotic and abiotic stresses and found that four AtVDAC transcripts were rapidly up-regulated in response to a bacterial pathogen.

To evaluate the involvement of translation initiation factors eIF4E and eIFiso4E in Chilli veinal mottle virus (ChiVMV) infection in pepper, we conducted a genetic analysis using a segregating population derived from a cross between Capsicum annuum 'Dempsey' containing an elF4E mutation ($pvr1^2$) and C. annuum 'Perennial' containing an elFiso4E mutation (pvr6). C. annuum 'Dempsey' was susceptible and C. annuum 'Perennial' was resistant to ChiVMV. All $F_1$ plants showed resistance, and $F_2$ individuals segregated in a resistant-susceptible ratio of 166:21, indicating that many resistance loci were involved. Seventy-five $F_2$ and 329 $F_3$ plants of 17 families were genotyped with $pvr1^2$ and pvr6 allele-specific markers, and the genotype data were compared with observed resistance to viral infection. All plants containing homozygous genotypes of both $pvr1^2$ and pvr6 were resistant to ChiVMV, demonstrating that simultaneous mutations in elF4E and eIFiso4E confer resistance to ChiVMV in pepper. Genotype analysis of $F_2$ plants revealed that all plants containing homozygous genotypes of both $pvr1^2$ and pvr6 showed resistance to ChiVMV. In protein-protein interaction experiments, ChiVMV viral genome-linked protein (VPg) interacted with both eIF4E and eIFiso4E. Silencing of elF4E and eIFiso4E in the VIGS experiment showed reduction in ChiVMV accumulation. These results demonstrated that ChiVMV can use both eIF4E and eIFiso4E for replication, making simultaneous mutations in eIF4E and eIFiso4E necessary to prevent ChiVMV infection in pepper.

We developed a novel on-chip activity assay using protein arrays for quantitative and rapid analysis of transglutaminase activity in mammalian cells. Transglutaminases are a family of $Ca^{2+}$-dependent enzymes involved in cell regulation as well as human diseases such as neurodegenerative disorders, inflammatory diseases and tumor progression. We fabricated the protein arrays by immobilizing N,N'-dimethylcasein (a substrate) on the amine surface of the arrays. We initiated transamidating reaction on the protein arrays and determined the transglutaminase activity by analyzing the fluorescence intensity of biotinylated casein. The on-chip transglutaminase activity assay was proved to be much more sensitive than the $[^3H]putrescine$-incorporation assay. We successfully applied the on-chip assay to a rapid and quantitative analysis of the transglutaminase activity in all-trans retinoic acid-treated NIH 3T3 and SH-SY5Y cells. In addition, the on-chip transglutaminase activity assay was sufficiently sensitive to determine the transglutaminase activity in eleven mammalian cell lines. Thus, this novel on-chip transglutaminase activity assay was confirmed to be a sensitive and high-throughput approach to investigating the roles of transglutaminase in cellular signaling, and, moreover, it is likely to have a strong potential for monitoring human diseases.

We previously reported that flavone induces embryonic lethality in Caenorhabditis elegans, which appeared to be the result of cell cycle arrest during early embryogenesis. To test this possibility, here we examined whether flavone inhibits the activity of a key cell cycle regulator, CDC-25.1 in C. elegans. A gain-of-function cdc-25.1 mutant, rr31, which exhibits extra cell divisions in intestinal cells, was used to test the inhibitory effects of flavone on CDC-25 activity. Flavone inhibited the extra cell divisions of intestinal cells in rr31, and modifications of flavone reduced the inhibitory effects. The inhibitory effects of flavone on CDC-25.1 were partly, if not completely, due to transcriptional repression.

Phytoestrogens are the natural compounds isolated from plants, which are structurally similar to animal estrogen, $17{\beta}$-estradiol. Tectoridin, a major isoflavone isolated from the rhizome of Belamcanda chinensis. Tectoridin is known as a phytoestrogen, however, the molecular mechanisms underlying its estrogenic effect are remained unclear. In this study we investigated the estrogenic signaling triggered by tectoridin as compared to a famous phytoestrogen, genistein in MCF-7 human breast cancer cells. Tectoridin scarcely binds to ER ${\alpha}$ as compared to $17{\beta}$-estradiol and genistein. Despite poor binding to ER ${\alpha}$, tectoridin induced potent estrogenic effects, namely recovery of the population of cells in the S-phase after serum starvation, transactivation of the estrogen response element, and induction of MCF-7 cell proliferation. The tectoridin-induced estrogenic effect was severely abrogated by treatment with U0126, a specific MEK1/2 inhibitor. Tectoridin promoted phosphorylation of ERK1/2, but did not affect phosphorylation of ER ${\alpha}$ at $Ser^{118}$. It also increased cellular accumulation of cAMP, a hallmark of GPR30-mediated estrogen signaling. These data imply that tectoridin exerts its estrogenic effect mainly via the GPR30 and ERK-mediated rapid nongenomic estrogen signaling pathway. This property of tectoridin sets it aside from genistein where it exerts the estrogenic effects via both an ER-dependent genomic pathway and a GPR30-dependent nongenomic pathway.

Chfr, a checkpoint with FHA and RING finger domains, plays an important role in cell cycle progression and tumor suppression. Chfr possesses the E3 ubiquitin ligase activity and stimulates the formation of polyubiquitin chains by Ub-conjugating enzymes, and induces the proteasome-dependent degradation of a number of cellular proteins, including Plk1 and Aurora A. While Chfr is a nuclear protein that functions within the cell nucleus, how Chfr is localized in the nucleus has not been clearly demonstrated. Here, we show that nuclear localization of Chfr is mediated by nuclear localization signal (NLS) sequences. To reveal the signal sequences responsible for nuclear localization, a short lysine-rich stretch (KKK) at amino acid residues 257-259 was replaced with alanine, which completely abolished nuclear localization. Moreover, we show that nuclear localization of Chfr is essential for its checkpoint function but not for its stability. Thus, our results suggest that NLS-mediated nuclear localization of Chfr leads to its accumulation within the nucleus, which may be important in the regulation of Chfr activation and Chfr-mediated cellular processes, including cell cycle progression and tumor suppression.

The chloroplast DNA sequences of Megaleranthis saniculifolia, an endemic and monotypic endangered plant species, were completed in this study (GenBank FJ597983). The genome is 159,924 bp in length. It harbors a pair of IR regions consisting of 26,608 bp each. The lengths of the LSC and SSC regions are 88,326 bp and 18,382 bp, respectively. The structural organizations, gene and intron contents, gene orders, AT contents, codon usages, and transcription units of the Megaleranthis chloroplast genome are similar to those of typical land plant cp DNAs. However, the detailed features of Megaleranthis chloroplast genomes are substantially different from that of Ranunculus, which belongs to the same family, the Ranunculaceae. First, the Megaleranthis cp DNA was 4,797 bp longer than that of Ranunculus due to an expanded IR region into the SSC region and duplicated sequence elements in several spacer regions of the Megaleranthis cp genome. Second, the chloroplast genomes of Megaleranthis and Ranunculus evidence 5.6% sequence divergence in the coding regions, 8.9% sequence divergence in the intron regions, and 18.7% sequence divergence in the intergenic spacer regions, respectively. In both the coding and noncoding regions, average nucleotide substitution rates differed markedly, depending on the genome position. Our data strongly implicate the positional effects of the evolutionary modes of chloroplast genes. The genes evidencing higher levels of base substitutions also have higher incidences of indel mutations and low Ka/Ks ratios. A total of 54 simple sequence repeat loci were identified from the Megaleranthis cp genome. The existence of rich cp SSR loci in the Megaleranthis cp genome provides a rare opportunity to study the population genetic structures of this endangered species. Our phylogenetic trees based on the two independent markers, the nuclear ITS and chloroplast MatK sequences, strongly support the inclusion of the Megaleranthis to the Trollius. Therefore, our molecular trees support Ohwi's original treatment of Megaleranthis saniculifolia to Trollius chosenensis Ohwi.

To investigate the regulatory mechanism underlying the contractile response in the intestinal smooth muscle of the nile tilapia (Orechromis niloticus), we used pharmacologic and molecular approaches to identify the muscarinic subreceptors and the intracellular signaling pathways involved in this motility. Myography assays revealed that an M1- and M3-subtype selective antagonist, but not a M2-subtype selective antagonist, inhibited carbachol HCl (CCH)-induced intestinal smooth muscle contraction. In addition, a phospholipase C inhibitor, but not an adenylate cyclase inhibitor, blocked the contractile response to CCH. We also cloned five muscarinic genes (OnM2A, OnM2B, OnM3, OnM5A, and OnM5B) from the nile tilapia. In the phylogenetic analysis and sequence comparison to compare our putative gene products (OnMs) with the sequences obtained from the near complete teleost genomes, we unexpectedly found that the teleost fish have respectively two paralogous genes corresponding to each muscarinic subreceptor, and other teleost fish, except zebrafish, do not possess muscarinic subreceptor M1. In addition, the expression pattern of the nile tilapia muscarinic subreceptor transcripts during CCH-induced intestinal smooth muscle contraction in the proximal intestinal tissue was analyzed by real-time PCR surveys and it was demonstrated that CCH increased the OnMs mRNA expression rapidly and transiently.