• Journal of Applied Biological Chemistry
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• 제48권3호
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• pp.120-126
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• 2005

Endothelial nitric oxide synthase (eNOS) plays a critical role in vascular biology and pathophysiology. Its activity is regulated by multiple mechanisms such as calcium/calmodulin, protein-protein interactions, sub-cellular locations and phosphorylation at various sites. Phosphorylation of eNOS-Ser1177 (based on mouse sequence) has been identified as an important mechanism of eNOS activation. However, signaling pathway leading to it phosphorylation remains controversial. The regulation of eNOS-Ser1177 phosphorylation by Src and protein kinase A (PKA) was investigated in the present study using cultured mouse aorta endothelial cells. Expression of a constitutively active Src mutant in the cells enhanced phosphorylation of eNOS and protein kinase B (Akt). The Src-stimulated phosphorylation was not attenuated by the expression of a dominant negative PKA regulatory subunit. Neither activation nor inhibition of PKA activity had any significant effect on tyrosine phosphorylation of activation or inactivation site in Src. Based on the results of this study, it is suggested that Src/Akt pathway and PKA signaling may regulate eNOS phosphorylation independently. The existence of multiple mechanisms for eNOS phosphorylation may guarantee endothelial nitric oxide production in various cellular contexts which is essential for maintenance of vascular health.

• Molecules and Cells
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• 제21권3호
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• pp.389-394
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• 2006

Human seven-in-absentia (SIAH)-interacting protein (SIP) is a component of the E3 ligase complex targeting beta-catenin for destruction. Arabidopsis has one SIP protein (AtSIP) with 32% amino acid sequence identity to SIP. To investigate the functions of AtSIP, we isolated an atsip knockout mutant, and generated transgenic plants overexpressing AtSIP. The growth rates and morphologies of the atsip and transgenic plants were indistinguishable from those of wild type. However, atsip plants were more susceptible to Pseudomonas syringae infection, and the transgenic plants overexpressing AtSIP were more resistant. Consistent with this, RNA blot analysis showed that the AtSIP gene is strongly induced by wounding and hydrogen peroxide treatment. In addition, when plants were infected with P. syringae, AtSIP was transiently induced prior to PR-1 induction. These observations show that Arabidopsis AtSIP plays a role in resistance to pathogenic infection.

• Natural Product Sciences
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• 제22권3호
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• pp.209-215
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• 2016

Kigelia africana (Lam.) Benth. (Bignoniaceae) is a flowering plants in South, Central and West Africa and commonly known as the sausage tree (Eng.); worsboom (Afr.); umVunguta, umFongothi (Zulu); Modukguhlu (North Sotho); Muvevha (Venda). The dried, powdered fruits are used as dressing for wounds and ulcers, haemorrhoids, rheumatism, purgative, skin-firming, lactation in breast-feeding mothers. The aim of this study is to investigate the cytotoxic and apoptotic potentials of 70% ethanolic extracts of Kigelia africana fruits in HCT116 human colon cancer cells. Treatment of Kigelia africana fruits with various concentrations resulted in a sequence of characteristic of apoptosis, including loss of cell viability and morphological changes. Flow cytometry analysis showed Kigelia africana fruits increased the sub-G1 phase (apoptosis) population. Apoptosis confirmed by annexin V-fluorescein isothiocyanate and propidium iodide double staining in HCT116 human colon cancer cell lines. Moreover, analysis of the mechanism indicated that Kigelia africana fruits showed an increased Bax and Bcl-2 expressions in a dose-dependent manner, resulting in activation of hallmarks of apoptotic events, caspase-3, caspase-9 and cleaved poly-ADP-ribose polymerase. This is the first report to demonstrate the cytotoxicity of Kigelia africana fruits on HCT116 human colon cancer cells.

• Biomolecules & Therapeutics
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• 제26권2호
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• pp.101-108
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• 2018

G protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane receptors and have vital signaling functions in various organs. Because of their critical roles in physiology and pathology, GPCRs are the most commonly used therapeutic target. It has been suggested that GPCRs undergo massive genetic variations such as genetic polymorphisms and DNA insertions or deletions. Among these genetic variations, non-synonymous natural variations change the amino acid sequence and could thus alter GPCR functions such as expression, localization, signaling, and ligand binding, which may be involved in disease development and altered responses to GPCR-targeting drugs. Despite the clinical importance of GPCRs, studies on the genotype-phenotype relationship of GPCR natural variants have been limited to a few GPCRs such as b-adrenergic receptors and opioid receptors. Comprehensive understanding of non-synonymous natural variations within GPCRs would help to predict the unknown genotype-phenotype relationship and yet-to-be-discovered natural variants. Here, we analyzed the non-synonymous natural variants of all non-olfactory GPCRs available from a public database, UniProt. The results suggest that non-synonymous natural variations occur extensively within the GPCR superfamily especially in the N-terminus and transmembrane domains. Within the transmembrane domains, natural variations observed more frequently in the conserved residues, which leads to disruption of the receptor function. Our analysis also suggests that only few non-synonymous natural variations have been studied in efforts to link the variations with functional consequences.

• Plant Biotechnology Reports
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• 제3권2호
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• pp.111-126
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• 2009

MicroRNAs (miRNAs) are endogenous, non-coding, small RNA molecules consisting of 21-24 nucleotides (nts) that regulate target genes at the posttranscriptional level in plants and animals. In plants, miRNAs negatively regulate target mRNAs containing a highly complementary sequence by either mRNA cleavage or translational repression. MiRNAs are processed from single-stranded precursors containing stem-loop structures by a Dicer-like enzyme and are loaded into silencing complexes, where they act on target mRNAs. Although plant miRNAs were first reported in Arabidopsis 10 years later than animal miRNAs, numerous miRNAs have since been identified from various land plants ranging from mosses to flowering plants, and their roles in diverse aspects of plant developmental processes have been characterized. Furthermore, most of the annotated plant miRNAs are evolutionarily conserved in various plants. In particular, recent functional studies using Arabidopsis mutants have contributed a great deal of information towards establishing a framework for understanding miRNA biogenesis and functional roles. Extensive appraisal of miRNA-directed regulation during a wide array of plant development and plant responses to environmental conditions has confirmed the versatile roles of miRNAs as a key component of plant molecular biology.

• Genomics & Informatics
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• 제13권3호
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• pp.76-80
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• 2015

Type 2 diabetes mellitus is a complex metabolic disorder associated with multiple genetic, developmental and environmental factors. The recent advances in gene expression microarray technologies as well as network-based analysis methodologies provide groundbreaking opportunities to study type 2 diabetes mellitus. In the present study, we used previously published gene expression microarray datasets of human skeletal muscle samples collected from 20 insulin sensitive individuals before and after insulin treatment in order to construct insulin-mediated regulatory network. Based on a motif discovery method implemented by iRegulon, a Cytoscape app, we identified 25 candidate regulons, motifs of which were enriched among the promoters of 478 up-regulated genes and 82 down-regulated genes. We then looked for a hierarchical network of the candidate regulators, in such a way that the conditional combination of their expression changes may explain those of their target genes. Using Genomica, a software tool for regulatory network construction, we obtained a hierarchical network of eight regulons that were used to map insulin downstream signaling network. Taken together, the results illustrate the benefits of combining completely different methods such as motif-based regulatory factor discovery and expression level-based construction of regulatory network of their target genes in understanding insulin induced biological processes and signaling pathways.

• Bulletin of the Korean Chemical Society
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• 제27권1호
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• pp.87-92
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• 2006

Fas-associated death domain protein (FADD) recruits and activates procaspase-8 through interactions between the death effector domains of these two proteins. Cellular FLICE-inhibitory protein (c-FLIP) was identified as a molecule with sequence homology to caspase-8. It has been postulated that c-FLIP prevents formation of the competent death-inducing signaling complex in a ligand-dependent manner, through its interaction with FADD and/or caspase-8. However, the interaction of FADD and $c-FLIP_s$ (short form) in apoptosis signaling has been controversially discussed. We show the purification and the characterization of human full-length FADD and $c-FLIP_s$ expressed in Escherichia coli. The purified FADD and $c-FLIP_s$ are shown as homogeneity, respectively, in SDS-PAGE analysis and light-scattering measurements. The folding properties of the $\alpha$-helical structure of FADD and the super-secondary structure of $c-FLIP_s$ proteins were characterized by circular dichroism spectroscopy. Furthermore, we report here a series of biochemical and biophysical data for FADD-$c-FLIP_s$ binding in vitro. The binding of both FADD and $c-FLIP_s$ proteins was detected by BIAcore biosensor, fluorescence measurement, and size-exclusion column (SEC).

• Molecules and Cells
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• 제37권4호
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• pp.295-301
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• 2014

SIFamide receptor (SIFR) is a Drosophila G protein-coupled receptor for the neuropeptide SIFamide (SIFa). Although the sequence and spatial expression of SIFa are evolutionarily conserved among insect species, the physiological function of SIFa/SIFR signaling remains elusive. Here, we provide genetic evidence that SIFa and SIFR promote sleep in Drosophila. Either genetic ablation of SIFa-expressing neurons in the pars intercerebralis (PI) or pan-neuronal depletion of SIFa expression shortened baseline sleep and reduced sleep-bout length, suggesting that it caused sleep fragmentation. Consistently, RNA interference-mediated knockdown of SIFR expression caused short sleep phenotypes as observed in SIFa-ablated or depleted flies. Using a panel of neuron-specific Gal4 drivers, we further mapped SIFR effects to subsets of PI neurons. Taken together, these results reveal a novel physiological role of the neuropeptide SIFa/SIFR pathway to regulate sleep through sleep-promoting neural circuits in the PI of adult fly brains.

• International Journal of Industrial Entomology and Biomaterials
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• 제12권2호
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• pp.57-61
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• 2006

Suppressor of cytokine signaling (SOCS) is known to be as a negative feedback regulator in Janus kinase signal transducer and activator of transcription signaling. Highly conserved SOCS box domain was cloned from a Korean malaria vector, Anopheles sinensis. Sequence analysis indicates that it has identity to Anopheles gambiae (96%), Aedes aegypti (94%), Drosophila melanogaster (78%), Mus musculus (72%) and Homo sapiens (72%), respectively. Tissue specificity RT-PCR demonstrated that the expression level of AsSOCS transcript was high at abdomen, midgut, and ovary, whereas developmental expression patterns showed that the level of AsSOCS was high at egg, early pupae, and adult female. On the other hand, RT-PCR analysis after bacterial challenge showed that SOCS mRNA was strongly induced in larvae. In addition, it was also induced by various immune elicitors such as lipoteicoic acid, CpG-DNA, and laminarin. It seems that AsSOCS, repressor of JAK-STAT pathway, is highly conserved in mosquito, and may play an important role in mosquito innate immune response.

• Molecules and Cells
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• 제39권5호
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• pp.403-409
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• 2016

NME1 is a well-known metastasis suppressor which has been reported to be downregulated in some highly aggressive cancer cells. Although most studies have focused on NME1, the NME1 gene also encodes the protein (NME1L) containing N-terminal 25 extra amino acids by alternative splicing. According to previous studies, NME1L has potent anti-metastatic activity, in comparison with NME1, by interacting with $IKK{\beta}$ and regulating its activity. In the present study, we tried to define the role of the N-terminal 25 amino acids of NME1L in $NF-{\kappa}B$ activation signaling. Unfortunately, the sequence itself did not interact with $IKK{\beta}$, suggesting that it may be not enough to constitute the functional structure. Further construction of NME1L fragments and biochemical analysis revealed that N-terminal 84 residues constitute minimal structure for homodimerization, $IKK{\beta}$ interaction and regulation of $NF-{\kappa}B$ signaling. The inhibitory effect of the fragment on cancer cell migration and $NF-{\kappa}B$-stimulated gene expression was equivalent to that of whole NME1L. The data suggest that the N-terminal 84 residues may be a core region for the anti-metastatic activity of NME1L. Based on this result, further structural analysis of the binding between NME1L and $IKK{\beta}$ may help in understanding the anti-metastatic activity of NME1L and provide direction to NME1L and $IKK{\beta}$-related anti-cancer drug design.