• BMB Reports
• /
• v.42 no.8
• /
• pp.516-522
• /
• 2009

SH2D4A, comprising a single SH2 domain, is a novel protein of the SH2 signaling protein family. We have previously demonstrated SH2D4A is expressed ubiquitously in various tissues and is located in the cytoplasm. In this study we investigated the function of SH2D4A in human embryonic kidney (HEK) 293 cells using interaction analysis, cell proliferation assays, and kinase activity detection. SH2D4A was found to directly bind to estrogen receptor $\alpha$ (ER$\alpha$), and prevent the recruitment of phospholipase C-$\gamma$ (PLC-$\gamma$) to ER$\alpha$. Moreover, we observed its inhibitory effects on estrogen-induced cell proliferation, involving the protein kinase C (PKC) signaling pathway. Together, these findings suggested that SH2D4A inhibited cell proliferation by suppression of the ER$\alpha$/PLC-$\gamma$/PKC signaling pathway. SH2D4A may be useful for the development of a new anti-cancer drug acting as an ER signaling modulator.

• ALGAE
• /
• v.32 no.4
• /
• pp.359-377
• /
• 2017

Pyropia haitanensis (T. J. Chang et B. F. Zheng) N. Kikuchi et M. Miyata is one of the most commercially useful macroalgae cultivated in southeastern China. In red algae, the biosynthesis of terpenoids through 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway can produce a direct influence on the synthesis of many biologically important metabolites. In this study, two genes of cDNAs, 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and 1-deoxy-D-xylulose-5-phosphate reductase (DXR), which encoding the first two rate-limiting enzymes among MEP pathway were cloned from P. haitanensis. The cDNAs of P. haitanensis DXS (PhDXS) and DXR (PhDXR) both contained complete open reading frames encoding polypeptides of 764 and 426 amino acids residues, separately. The expression analysis showed that PhDXS was significant differently expressed between leafy thallus and conchocelis as PhDXR been non-significant. Additionally, expression of PhDXR and its downstream gene geranylgeranyl diphosphate synthase were both inhibited by fosmidomycin significantly. Meanwhile, we constructed types of phylogenetic trees through different algae and higher plants DXS and DXR encoding amino acid sequences, as a result we found tree clustering consequences basically in line with the "Cavalier-Smith endosymbiotic theory." Whereupon, we speculated that in red algae, there existed only complete MEP pathway to meet needs of terpenoids synthesis for themselves; Terpenoids synthesis of red algae derivatives through mevalonate pathway came from two or more times endosymbiosis of heterotrophic eukaryotic parasitifer. This study demonstrated that PhDXS and PhDXR could play significant roles in terpenoids biosynthesis at molecular levels. Meanwhile, as nuclear genes among MEP pathway, PhDXS and PhDXR could provide a new way of thinking to research the problem of chromalveolata biological evolution.

• The Journal of Korean Medicine
• /
• v.27 no.4
• /
• pp.74-83
• /
• 2006

Ssanghwatang (SHT) has been known to prove effective in the treatment for erectile dysfunction (ED), and its modified formula is widely used in clinical practice. However, its fundamental mechanism of action is not clearly known. It is well known that endothelial cells can achieve the relaxation of vascular smooth muscles by the release of nitric oxide (NO). NO is synthesized by the enzyme NO synthase (NOS) from L-arginine and oxygen. It is widely accepted that NO plays an important role in the relaxation of corpus cavernous smooth muscle and vasculature. In addition, in terms of the penile erection, the NO/cGMP pathway is more potent than the PCE1/cAMP pathway. The main purpose of the present study was to investigate the mechanism of the erectile effects of SHT by focusing on its direct effects on corpus cavernous smooth muscle cells. We investigated the NOS activity, nitrite concentration and cGMP levels in rat corpus cavernous smooth muscle cell lines activated by SHT extracts. Furthermore, we evaluated the effect of SHT extracts on penile smooth muscle relaxation following oral administration of SHT extract powder to rats by the dosage of 1 g/kg over fifteen days. As a result, we found that SHT stimulated NO release. NOS activity and cGMP levels were increased by SHT respectively. Furthermore, SHT relaxed the corpus cavernous smooth muscle. These results are consistent with the concept that penile erection by SHT is carried out through the NO/cGMP pathway. In conclusion, the present study shows that SHT increases the NOS activity, synthesizes NO and augments the cGMP, which mediates penile erection. Further determination of the SHT mechanism related with the NO/cGMP pathway strongly indicates that SHT can be used as a remedy for erectile impotence.

• Molecules and Cells
• /
• v.26 no.4
• /
• pp.344-349
• /
• 2008

5-Fluorouracil (5-FU), a pyrimidine antagonist, has a long history in cancer treatment. The targeted pyrimidine biosynthesis pathway includes dihydropyrimidine dehydrogenase (DPD), which converts 5-FU to an inactive metabolite, and thymidylate synthase (TS), which is a major target of 5-FU. Using Caenorhabditis elegans as a model system to study the functional and resistance mechanisms of anti-cancer drugs, we examined these two genes in order to determine the extent of molecular conservation between C. elegans and humans. Overexpression of the worm DPD and TS homologs (DPYD-1 and Y110A7A.4, respectively) suppressed germ cell death following 5-FU exposure. In addition, DPYD-1 depletion by RNAi resulted in 5-FU sensitivity, while treatment with Y110A7A.4 RNAi and 5-FU resulted in similar patterns of embryonic death. Thus, the pathway of 5-FU function appears to be highly conserved between C. elegans and humans at the molecular level.

• International Journal of Molecular Medicine
• /
• v.44 no.3
• /
• pp.1161-1171
• /
• 2019

The present study investigated whether glucagon like peptide-1 (GLP-1) improves glucose uptake through glucose transporter type 4 (GLUT4), mediated by the activation of sirtuin 1 (SIRT1), in skeletal muscle cells with palmitate induced-insulin resistance. The levels of glucose uptake, GLUT4, protein kinase A (PKA), and cyclic adenosine monophosphate (cAMP) were determined in human skeletal muscle myotubes (HSMMs) exposed to palmitate and GLP-1. Then, to determine whether PKA/cAMP were downstream signals of GLP-1, a PKA inhibitor was used. To determine whether SIRT-1 contributes to GLP-1 action in HSMMs with palmitate-induced insulin resistance, the levels of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) deacetylation and SIRT-1 activity were assessed using a SIRT1 inhibitor and small interfering RNA (siRNA). The phosphorylation levels of protein kinase B (Akt) and insulin receptor substrate 1 (IRS-1) as insulin signaling pathways, were assessed in GLP-1-treated HSMMs exposed to palmitate. The influence of SIRT1 on the GLP-1-induced activation of insulin signaling pathway was determined using a SIRT1 inhibitor. GLP-1 restored the palmitate-induced reductions in the levels of glucose uptake, GLUT4 mRNA, GLUT4 promoter activity, and GLUT4 protein in HSMMs. PKA and cAMP, as GLP-1 downstream signals, played a role in this process. GLP-1 increased the deacetylation levels of PGC1α, and stimulated SIRT1 in HSMMs. Moreover, the SIRT1 inhibitor and siRNA of SIRT1 suppressed the effect of GLP-1 on GLUT4 expression in HSMMs exposed to palmitate. The SIRT1 inhibitor also prevented the GLP-1-induced phosphorylation of IRS-1 and Akt in palmitate-treated HSMMs. The present findings suggest that in palmitate-induced insulin-resistant HSMM, GLP-1 activates SIRT1 through the PKA/cAMP pathway, which in turn enhances glucose uptake through GLUT4 and the insulin signaling pathway.

• The Korean Journal of Physiology and Pharmacology
• /
• v.4 no.6
• /
• pp.487-496
• /
• 2000

Insulin stimulates glucose transport in muscle and fat cells by promoting the translocation of glucose transporter (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3-kinase) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt and $PKC-{\zeta}$, those are known as the downstream target of PI3-kinase in regulation of GLUT4 translocation, is not known yet. An interesting possibility is that these protein kinases phosphorylate GLUT4 directly in this process. In the present study, $PKB-{\alpha}$ and $PKC-{\zeta}$ were added exogenously to GLUT4-containing vesicles purified from low density microsome (LDM) of the rat adipocytes by immunoadsorption and immunoprecipitation for direct phosphorylation of GLUT4. Interestingly GLUT4 was phosphorylated by $PKC-{\zeta}$ and its phosphorylation was increased in insulin stimulated state but GLUT4 was not phosphorylated by $PKB-{\alpha}.$ However, the GST-fusion proteins, GLUT4 C-terminal cytoplasmic domain (GLUT4C) and the entire major GLUT4 cytoplasmic domain corresponding to N-terminus, central loop and C-terminus in tandem (GLUT4NLC) were phosphorylated by both $PKB-{\alpha}$ and $PKC-{\zeta}.$ The immunoblots of $PKC-{\zeta}$ and $PKB-{\alpha}$ antibodies with GLUT4-containing vesicles preparation showed that $PKC-{\zeta}$ was co-localized with the vesicles but not $PKB-{\alpha}.$ From the above results, it is clear that $PKC-{\zeta}$ interacts with GLUT4-containing vesicles and it phosphorylates GLUT4 protein directly but $PKB-{\alpha}$ does not interact with GLUT4, suggesting that insulin-elicited signals that pass through PI3-kinase subsequently diverge into two independent pathways, an Akt pathway and a $PKC-{\zeta}$ pathway, and that later pathway contributes, at least in part, insulin stimulation of GLUT4 translocation in adipocytes via a direct GLUT4 phosphorylation.

• Journal of Acupuncture Research
• /
• v.18 no.4
• /
• pp.101-115
• /
• 2001

Objective : To characterize the antitumorigenic potential of Apamin, one of the major components of bee venom, its effects on cell proliferation and the mitogen-activated protein kinase (MAPK) signal transduction pathway were characterized using the human melanoma cell line SK-MEL-2. Methods & Results : Cell counting analysis for cell death demonstrated that consistent with a previous results, SK-MEL-2 cells treated with $0.5-2.0{\mu}g/ml$ of Apamin showed no recognizable cytotoxic effect whereas detectable induction of cell death was identified at concentrations over $5.0{\mu}g/ml$. [3H]thymidine incorporation assay for cell proliferation demonstrated that DNA replication of SK-MEL-2 cells is inhibited by Apamin in a dose- and time-dependent manner. To explore whether Apamin-induced growth suppression is associated with the MAPK signaling pathway, phosphorylation of Erk, a function mediator of MAPK growth-stimulating signal, was examined Western blot assay using a phospho-specific Erkl/2 antibody. A significant increase of Erkl/2 phosphorylation level was observed in Apamin-treated cells compared with untreated control cells. Qantitative RT-PCR analysis revealed that Apamin inhibit expression of MAPK downstream genes such as c-Jun, c-Fos, and cyclin D1 but not expression of MAPK pathway component genes including Ha-Ras, c-Raf-1, MEK1, and Erk. Conclusion : It is strongly suggested that the antitumorigenic activity of Apamin might result in part from its inhibitory effect on the MAPK signaling pathway in human melanoma cells SK-MEL-2.

• Applied Biological Chemistry
• /
• v.36 no.3
• /
• pp.184-190
• /
• 1993

The effect of ${\delta}-aminolevulinic\;acid$ (ALA) biosynthetic precursors and related compounds on the ALA productivity from a strain of Rhodobacter sphaeroides has been examined in vivo and in vitro systems. The relative ratios of ALA productivities by $C_{4}$- pathway to that by $C_{5}$-pathway in vivo and in vitro systems were 0.78 and 1.37, respectively. Although the expression rates of $C_{4}-$ and $C_{5}-pathways$ in cell-free systems prepared after precursors supplemented cultivations were increased 1.35 and 1.52 folds, respectively, the rate increase of $C_{4}-pathway$ was accompanied by the rate decrease of the $C_{5}-pathways$, and vice versa, as that the rates of both $C_{4}-$ and $C_{5}-pathways$ were lowered to be 0.91, 0.83, respectively. The order of cellular uptake rates of ${\gamma}-glutamyl$ derivatives relative to that found with L-glutamic acid were shown to be D-glutamic acid, 0.55: D-glutamine, 0.5: L-glutamine, 0.4: ${\gamma}-L-glutamyl$ ethylester, 0.3: GSH and Glu-pNA, 0. L and D configurations of glutamine were indicated as better substrates in vivo for ALA yields than those of glutamic acid, respectively.

• Journal of Integrative Natural Science
• /
• v.4 no.3
• /
• pp.202-209
• /
• 2011

Tuberculosis is a major health problem in humans because of its multidrug resistance and discovering new treatments for this disease is urgently required. The synthesis of isoprenoids in Mycobacterium tuberculosis has been reported as an interesting pathway to target. In this context, 2C-methyl-D-erythritol 4-phosphate (MEP) pathway of M. tuberculosis has drawn attention. The MEP pathway begins with the condensation of glyceraldehyde 3-phosphate and pyruvate forming 1-deoxy-D-xylulose 5-phosphate (DXP) which is catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS). As there is no X-ray structure was reported for this target, comparative modeling was used to generate the three dimensional structure. The structure was further validated by PROCHECK, VERIFY-3D, PROSA, ERRAT and WHATIF. Molecular docking studies was performed with the substrate (Thiamine pyrophosphate) and the reported inhibitor 2-methyl-3-(4-fluorophenyl)-5-(4-methoxy-phenyl)-4H-pyrazolol[1,5-a]pyrimidin-7-one) against the developed model to identify the crucial residues in the active site. This study may further be useful to provide structure based drug design.

• Microbiology and Biotechnology Letters
• /
• v.21 no.6
• /
• pp.527-533
• /
• 1993

Aminolevulinic acid(ALA) was shown to be synthesized via active pathways of either C4 or C5 ALA biosynthesis in cells of a photosynthetic bacterium, Rhodocyclus gelatinosus KUP-74, where the C5 pathway was appeared to be preferntially expressed in the cells. It was strongly suggested that L-glutamine might be utilized more effectively than L-glutamate to synthesize ALA via C5 pathway in this bacterium from the fact of relationship between the cellular uptake rates of glutamate and its Gamma-derivaties and corresponded ALA productivities in vitro and in vivo.