Young-Seuk Bae

Bone-Protecting Effect of Safflower Seeds in Ovariectomized Rats

Safflower (Carthamus tinctorius L.) seeds have long been clinically used in Korea to promote bone formation and prevent osteoporosis. However, the beneficial effect has not been scientifically evaluated. Thus, in the present study we investigated whether phytoestrogen rich safflower seeds reduce bone loss in ovariectomized rats. Female Sprague-Dawley rats were subjected to bilateral ovariectomy or sham surgery. One week after the operation, ovariectomized rats were either fed a diet containing defatted safflower seeds or injected with 17b-estradiol (E2) for 4 weeks. As expected, ovariectomy resulted in a dramatic reduction in trabecular bone mass of the proximal tibia, increase in deposition of marrow fat, and in uterine atrophy. E2 treatment almost completely prevented bone loss as well as marrow adiposity, as examined by scanning electron microscopy and histomorphometry. Safflower seeds partially prevented ovariectomy-induced bone loss and slightly reduced marrow adiposity. Safflower seeds, in contrast to E2, exerted very weak uterotrophic action. In an attempt to elucidate the underlying mechanisms, effect of polyphenolic compounds extracted from safflower seeds on proliferation of osteoblast-like cells was also assessed in vitro. The mixed polyphenolic compounds stimulated growth of ROS 17/2.8 osteoblast-like cells in a dose-dependent manner (5-100 mg/ml), as potently as E2 and genistein. The present data provide the first direct in vivo evidence that safflower seeds have a protecting effect on bone loss caused by estrogen deficiency, without substantial effect on the uterus. The beneficial effect of safflower seeds may be mediated, at least in part, by the stimulating effect of polyphenolic compounds on proliferation of osteoblasts.

Performance evaluation of thermal cyclers for PCR in a rapid cycling condition

The performance of thermal cyclers for polymerase chain reactions (PCR) is of great concern in terms of the reliability of PCR-based assays, particularly when rapid cycling conditions are applied to small volume reactions. In this work, the precision of the temperature controls during rapid thermal cycling was measured in 19 commercial thermal cyclers of 8 different models. The temperatures of test solutions in specific locations in each thermal block were simultaneously monitored at 1 s intervals during thermal cycling. A temperature-sensitive multiplex PCR was run in parallel to assess undesirable PCR results caused by poor temperature control. Under the given conditions (20 s of annealing time and 20 microL reaction volume), a majority of the tested instruments showed prominent curving, undershooting, and/or overshooting in their temperature profiles, which substantially influenced the results of the temperature-sensitive multiplex PCR. Variations between wells were also observed in most instruments. It is strongly hoped that these problems will be addressed by manufacturers and that they will make substantial improvements in the precision and efficiency of thermal cyclers. In the meantime, users of thermal cyclers might be able to avoid unexpected poor outcomes of sensitive PCR-based assays by designing their PCR protocols with these findings in mind.

Regulation of Telomeric Repeat Binding Factor 1 Binding to Telomeres by Casein Kinase 2-mediated Phosphorylation

Telomere maintenance is essential for continued cell proliferation and chromosome stability. Telomeres are maintained by telomerase and a collection of associated proteins. The telomeric protein telomeric repeat binding factor 1 (TRF1) negatively regulates telomere length by inhibiting access of telomerase at telomere termini. Here we report that TRF1 interacts with the β subunit of casein kinase 2 (CK2) and serves as a substrate for CK2. CK2-mediated phosphorylation is required for the efficient telomere binding of TRF1 in vitro and in vivo. Inhibition of CK2 by the CK2 inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole decreased the ability of TRF1 to bind telomeric DNA. The resulting telomere-unbound form of TRF1 was then ubiquitinated and degraded by the proteasome. Partial knockdown of CK2 by small interfering RNA resulted in removal of TRF1 from telomeres and subsequent degradation of TRF1. Mapping of the CK2 target site identified threonine 122 as a substrate in TRF1. A threonine to alanine change at this position led to a diminished DNA binding due to reduced dimerization of TRF1. In addition, phosphorylation of threonine 122 seemed critical for TRF1-mediated telomere length control. Our findings suggest that CK2-mediated phosphorylation of TRF1 plays an important role in modulating telomere length homeostasis by determining the levels of TRF1 at telomeres.

The p53-p21Cip1/WAF1 pathway is necessary for cellular senescence induced by the inhibition of protein kinase CKII in human colon cancer cells

We have previously shown that the down-regulation of protein kinase CKII activity is tightly associated with cellular senescence of human fibroblast IMR-90 cells. Here, we examined the roles of p53 and p21Cip1/WAF1 in senescence development induced by CKII inhibition using wild-type, isogenic p53-/- and isogenic p21-/- HCT116 human colon cancer cell lines. A senescent marker appeared after staining for senescence-associated β-galactosidase activity in wild-type HCT116 cells treated with CKII inhibitor or CKIIα siRNA, but this response was almost abolished in p53- or p21Cip1/WAF1-null cells. Increased cellular levels of p53 and p21Cip1/WAF1 protein occurred with the inhibition of CKII. CKII inhibition upregulated p53 and p21Cip1/WAF1 expression at post-transcriptional level and transcription level, respectively. RB phosphorylation significantly decreased in cells treated with CKII inhibitor. Taken together, this study shows that the activation of the p53–p21Cip1/WAF1 pathway acts as a major mediator of cellular senescence induced by CKII inhibition.

Melittin suppresses EGF-induced cell motility and invasion by inhibiting PI3K/Akt/mTOR signaling pathway in breast cancer cells.

Bee venom is a natural compound produced by the honey bee (Apis mellifera), and has been reported as having the biological and pharmacological activities, including anti-bacterial, anti-viral and anti-inflammation. In the present study, the inhibitory effects of bee venom and its major peptide components on the tumor invasion were demonstrated. It was confirmed the inhibitory effects of bee venom, melittin, and apamin on the EGF-induced invasion of breast cancer cells. Transwell invasion and wound-healing assays showed that bee venom and melittin significantly inhibits the EGF-induced invasion and migration of breast cancer cells. Also, bee venom and melittin reduced the EGF-stimulated F-actin reorganization at the leading edge, but apamin did not affect. Particularly, melittin inhibited the EGF-induced MMP-9 expression via blocking the NF-κB and PI3K/Akt/mTOR pathway. In addition, melittin significantly suppressed the EGF-induced FAK phosphorylation through inhibition of mTOR/p70S6K/4E-BP1 pathway. These results suggest that inhibitory effects of melittin on breast cancer cell motility and migration may be related to the inhibition of mTOR pathway.

Development of FET-type albumin sensor for diagnosing nephritis.

An albumin biosensor based on a potentiometric measurement using Biofield-effect-transistor (BioFET) has been designed and fabricated, and its characteristics were investigated. The BioFET was fabricated using semiconductor integrated circuit (IC) technology. The gate surface of the BioFET was chemically modified by newly developed self-assembled monolayer (SAM) synthesized by a thiazole benzo crown ether ethylamine (TBCEA)-thioctic acid to immobilize anti-albumin. SAM formation, antibody immobilization, and antigen-antibody interaction were verified using surface plasmon resonance (SPR). The output voltage changes of the BioFET with respect to various albumin concentrations were obtained. Quasi-reference electrode (QRE) and reference FET (ReFET) has been integrated with the BioFET, and its output characteristic was investigated. The results demonstrate the feasibility of the BioFET as the albumin sensor for diagnosing nephritis.

Phospholipase D is involved in oxidative stress-induced migration of vascular smooth muscle cells via tyrosine phosphorylation and protein kinase C

Oxidative stress has been implicated in mediation of vascular disorders. In the presence of vanadate, H2O2 induced tyrosine phosphorylation of PLD1, protein kinase C-α (PKC-α), and other unidentified proteins in rat vascular smooth muscle cells (VSMCs). Interestingly, PLD1 was found to be constitutively associated with PKC-α in VSMCs. Stimulation of the cells by H2O2 and vanadate showed a concentration-dependent tyrosine phosphorylation of the proteins in PLD1 immunoprecipitates and activation of PLD. Pretreatment of the cells with the protein tyrosine kinase inhibitor, genistein resulted in a dose-dependent inhibition of H2O2-induced PLD activation. PKC inhibitor and down-regulation of PKC abolished H2O2-stimulated PLD activation. The cells stimulated by oxidative stress (H2O2) caused increased cell migration. This effect was prevented by the pretreatment of cells with tyrosine kinase inhibitors, PKC inhibitors, and 1-butanol, but not 3-butanol. Taken together, these results suggest that PLD might be involved in oxidative stress-induced migration of VSMCs, possibly via tyrosine phosphorylation and PKC activation.

Upregulation of miR-760 and miR-186 Is Associated with Replicative Senescence in Human Lung Fibroblast Cells

We have previously shown that microRNAs (miRNAs) miR-760, miR-186, miR-337-3p, and miR-216b stimulate premature senescence through protein kinase CK2 (CK2) down-regulation in human colon cancer cells. Here, we examined whether these four miRNAs are involved in the replicative senescence of human lung fibroblast IMR-90 cells. miR-760 and miR-186 were significantly upregulated in replicatively senescent IMR-90 cells, and their joint action with both miR-337-3p and miR-216b was necessary for efficient downregulation of the α subunit of CK2 (CK2α) in IMR-90 cells. A mutation in any of the four miRNA-binding sequences within the CK2α 3′-untranslated region (UTR) indicated that all four miRNAs should simultaneously bind to the target sites for CK2α downregulation. The four miRNAs increased senescence-associated β-galactosidase (SA-β-gal) staining, p53 and p21Cip1/WAF1 expression, and reactive oxygen species (ROS) production in proliferating IMR-90 cells. CK2α over-expression almost abolished this event. Taken together, the present results suggest that the upregulation of miR-760 and miR-186 is associated with replicative senescence in human lung fibroblast cells, and their cooperative action with miR-337-3p and miR-216b may induce replicative senescence through CK2α downregulation-dependent ROS generation.

In Vivo AAV1 Transduction With hRheb(S16H) Protects Hippocampal Neurons by BDNF Production

Recent evidence has shown that Ras homolog enriched in brain (Rheb) is dysregulated in Alzheimers disease (AD) brains. However, it is still unclear whether Rheb activation contributes to the survival and protection of hippocampal neurons in the adult brain. To assess the effects of active Rheb in hippocampal neurons in vivo, we transfected neurons in the cornu ammonis 1 (CA1) region in normal adult rats with an adeno-associated virus containing the constitutively active human Rheb (hRheb(S16H)) and evaluated the effects on thrombin-induced neurotoxicity. Transduction with hRheb(S16H) significantly induced neurotrophic effects in hippocampal neurons through activation of mammalian target of rapamycin complex 1 (mTORC1) without side effects such as long-term potentiation impairment and seizures from the alteration of cytoarchitecture, and the expression of hRheb(S16H) prevented thrombin-induced neurodegeneration in vivo, an effect that was diminished by treatment with specific neutralizing antibodies against brain-derived neurotrophic factor (BDNF). In addition, our results showed that the basal mTORC1 activity might be insufficient to mediate the level of BDNF expression, but hRheb(S16H)-activated mTORC1 stimulated BDNF production in hippocampal neurons. These results suggest that viral vector transduction with hRheb(S16H) may have therapeutic value in the treatment of neurodegenerative diseases such as AD.

Phospholipase D2 downregulation induces cellular senescence through a reactive oxygen species–p53–p21Cip1/WAF1 pathway

The expression of phospholipase D1 (PLD1) and PLD2 were found to decrease at the transcription level during both replicative and premature senescence in human lung fibroblast IMR‐90 cells. Knockdown of PLD2 dramatically induced senescent phenotype in proliferating IMR‐90 cells and wild‐type HCT116 colon cancer cells, whereas this response was nearly abolished in p53‐ or p21Cip1/WAF1‐null HCT116 cells. PLD2 knockdown increased the intracellular reactive oxygen species (ROS). Antioxidant N‐acetyl‐l‐cysteine, NADPH oxidase inhibitor apocynin, and p22phox small interfering RNA (siRNA) reduced ROS generation and thus suppressed the appearance of senescence markers. Elevated CK2 α subunit (CK2α) expression repressed PLD2 downregulation‐mediated senescence. PLD2 overexpression increased protein kinase CK2 (also known as casein kinase 2) (CK2) activity. Taken together, these results show that PLD2 downregulation causes senescence through the p53–p21Cip1/WAF1 pathway by stimulating ROS production, which is induced by CK2 inhibition.