USP39 promotes non-homologous end-joining repair by poly(ADP-ribose)-induced liquid demixing.

Mutual crosstalk among poly(ADP-ribose) (PAR), activated PAR polymerase 1 (PARP1) metabolites, and DNA repair machinery has emerged as a key regulatory mechanism of the DNA damage response (DDR). However, there is no conclusive evidence of how PAR precisely controls DDR. Herein, six deubiquitinating enzymes (DUBs) associated with PAR-coupled DDR were identified, and the role of USP39, an inactive DUB involved in spliceosome assembly, was characterized. USP39 rapidly localizes to DNA lesions in a PAR-dependent manner, where it regulates non-homologous end-joining (NHEJ) via a tripartite RG motif located in the N-terminus comprising 46 amino acids (N46). Furthermore, USP39 acts as a molecular trigger for liquid demixing in a PAR-coupled N46-dependent manner, thereby directly interacting with the XRCC4/LIG4 complex during NHEJ. In parallel, the USP39-associated spliceosome complex controls homologous recombination repair in a PAR-independent manner. These findings provide mechanistic insights into how PAR chains precisely control DNA repair processes in the DDR.

Staufen1-mediated mRNA decay induces Requiem mRNA decay through binding of Staufen1 to the Requiem 3'UTR.

Requiem (REQ/DPF2) was originally identified as an apoptosis-inducing protein in mouse myeloid cells and belongs to the novel Krüppel-type zinc finger d4-protein family of proteins, which includes neuro-d4 (DPF1) and cer-d4 (DPF3). Interestingly, when a portion of the REQ messenger ribonucleic acid (mRNA) 3' untranslated region (3'UTR), referred to as G8, was overexpressed in K562 cells, ß-globin expression was induced, suggesting that the 3'UTR of REQ mRNA plays a physiological role. Here, we present evidence that the REQ mRNA 3'UTR, along with its trans-acting factor, Staufen1 (STAU1), is able to reduce the level of REQ mRNA via STAU1-mediated mRNA decay (SMD). By screening a complementary deoxyribonucleic acid (cDNA) expression library with an RNA-ligand binding assay, we identified STAU1 as an interactor of the REQ mRNA 3'UTR. Specifically, we provide evidence that STAU1 binds to putative 30-nucleotide stem-loop-structured RNA sequences within the G8 region, which we term the protein binding site core; this binding triggers the degradation of REQ mRNA and thus regulates translation. Furthermore, we demonstrate that siRNA-mediated silencing of either STAU1 or UPF1 increases the abundance of cellular REQ mRNA and, consequently, the REQ protein, indicating that REQ mRNA is a target of SMD.

Low concentrations of doxycycline attenuates FasL-induced apoptosis in HeLa cells.

BACKGROUND: Doxycycline (DC) has been shown to possess non-antibiotic properties including Fas/Fas Ligand (FasL)-mediated apoptosis against several tumor types in the concentration range of 10-40 µg/mL. However, the effect of DC in apoptotic signaling at much low concentrations was not studied. METHODS: The present study investigated the attenuation effect of low dose of DC on FasL-induced apoptosis in HeLa cell by the methods of MTT assay, fluorescence microscopy, DNA fragmentation, flow cytometry analysis, and western blotting. RESULTS AND CONCLUSION: In the present findings we showed that low concentration of DC (<2.0 µg/mL) exhibited protective effects against FasL-induced apoptosis in HeLa cells. FasL treatment to HeLa cells resulted in a concentration-dependent induction of cell death, and treatment with low concentrations of DC (0.1-2 µg/mL) significantly (p < 0.001) attenuated the FasL-induced cell death as measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Further, the FasL-induced apoptotic features in HeLa cells, such as morphological changes, DNA fragmentation and cell cycle arrest was also inhibited by DC (0.5 µg/mL). Tetracycline and minocycline also showed similar anti-apoptotic effects but were not significant when compared to DC, tested at same concentrations. Further, DC (0.01-16 µg/mL) did not influence the hydrogen peroxide- or cisplatin-induced intrinsic apoptotic pathway in HeLa cells. Protein analysis using Western blotting confirmed that FasL-induced cleavage/activation of caspase-8 and caspase-3, were inhibited by DC treatment at low concentration (0.5 µg/mL). Considering the overall data, we report for the first time that DC exhibited anti-apoptotic effects at low concentrations in HeLa cells by inhibition of caspase activation via FasL-induced extrinsic pathway.

Development of an Anti-HER2 Single-Chain Variable Antibody Fragment Construct for High-Yield Soluble Expression in Escherichia coli and One-Step Chromatographic Purification.

Although single-chain variable fragment (scFv) is recognized as a highly versatile scaffold of recombinant antibody fragment molecules, its overexpression in Escherichia coli often leads to the formation of inclusion bodies. To address this issue, we devised and tested four different constructs, named v21, v22, v23 and v24, for producing anti-human epidermal growth factor receptor 2 (HER2) scFv. Among them, the v24 construct obtained from N-terminal fusion of maltose-binding protein (MBP) and subsequent tobacco etch virus protease (TEV) was identified as the most efficient construct for the production of anti-HER2 scFv. Aided by an MBP tag, high-yield soluble expression was ensured and soluble scFv was liberated in cells via autonomous proteolytic cleavage by endogenously expressed TEV. The isolated scFv containing a C-terminal hexahistidine tag was purified through a one-step purification via nickel-affinity chromatography. The purified scFv exhibited a strong (nanomolar Kd) affinity to HER2 both in vitro and in cells. Structural and functional stabilities of the scFv during storage for more than one month were also assured. Given the great utility of anti-HER2 scFv as a basic platform for developing therapeutic and diagnostic agents for cancers, the v24 construct and methods presented in this study are expected to provide a better manufacturing system for producing anti-HER2 scFv with various industrial applications.

A Cell-Penetrant Peptide Disrupting the Transcription Factor CP2c Complexes Induces Cancer-Specific Synthetic Lethality.

Despite advances in precision oncology, cancer remains a global public health issue. In this report, proof-of-principle evidence is presented that a cell-penetrable peptide (ACP52C) dissociates transcription factor CP2c complexes and induces apoptosis in most CP2c oncogene-addicted cancer cells through transcription activity-independent mechanisms. CP2cs dissociated from complexes directly interact with and degrade YY1, leading to apoptosis via the MDM2-p53 pathway. The liberated CP2cs also inhibit TDP2, causing intrinsic genome-wide DNA strand breaks and subsequent catastrophic DNA damage responses. These two mechanisms are independent of cancer driver mutations but are hindered by high MDM2 p60 expression. However, resistance to ACP52C mediated by MDM2 p60 can be sensitized by CASP2 inhibition. Additionally, derivatives of ACP52C conjugated with fatty acid alone or with a CASP2 inhibiting peptide show improved pharmacokinetics and reduced cancer burden, even in ACP52C-resistant cancers. This study enhances the understanding of ACP52C-induced cancer-specific apoptosis induction and supports the use of ACP52C in anticancer drug development.

PIAS1 regulates CP2c localization and active promoter complex formation in erythroid cell-specific alpha-globin expression.

Data presented here extends our previous observations on α-globin transcriptional regulation by the CP2 and PIAS1 proteins. Using RNAi knockdown, we have now shown that CP2b, CP2c and PIAS1 are each necessary for synergistic activation of endogenous α-globin gene expression in differentiating MEL cells. In this system, truncated PIAS1 mutants lacking the ring finger domain recruited CP2c to the nucleus, as did wild-type PIAS1, demonstrating that this is a sumoylation-independent process. In vitro, recombinant CP2c, CP2b and PIAS1 bound DNA as a stable CBP (CP2c/CP2b/PIAS1) complex. Following PIAS1 knockdown in MEL cells, however, the association of endogenous CP2c and CP2b with the α-globin promoter simultaneously decreased. By mapping the CP2b- and CP2c-binding domains on PIAS1, and the PIAS1-binding domains on CP2b and CP2c, we found that two regions of PIAS1 that interact with CP2c/CP2b are required for its co-activator function. We propose that CP2c, CP2b, and PIAS1 form a hexametric complex with two units each of CP2c, CP2b, and PIAS1, in which PIAS1 serves as a clamp between two CP2 proteins, while CP2c binds directly to the target DNA and CP2b mediates strong transactivation.

Mechanism of activation of human c-KIT kinase by internal tandem duplications of the juxtamembrane domain and point mutations at aspartic acid 816.

The proto-oncogene c-KIT receptor has been implicated as an essential component in the activation of leukemic cells. The internal tandem duplication (ITD) of c-KIT has also been identified as a predominant cause of acute myeloid leukemia (AML), although its role in the activation process is still unclear. To investigate the biological mechanisms of c-KIT activation, we generated a c-KIT receptor bearing two different immunological tags, HA and Flag tags. In this study, we demonstrated that the mutant (Mt)-ITD and Asp816 (D816Y) c-KIT receptors spontaneously formed dimers and that these Mt-ITD forms of c-KIT displayed high levels of phosphorylation and increased cellular tyrosine phosphorylation. The amount of wild-type homodimers increased following the addition of the c-KIT ligand, while the level of mutant homodimers was less affected by the addition of the c-KIT ligand. Furthermore, we demonstrated that Mt-ITD and activating point mutations of D816Y induced constitutive activation of c-KIT kinase in the absence of ligand in COS-1 cells. These data suggest a novel mechanism for the regulation of cell growth autonomy. Overall, our study suggests that c-KIT activation might have significant effects on hematopoietic cells and might help to improve our understanding of the pathogenesis of systemic mast cell disease, gastrointestinal stromal tumors and AML and potentially lead to the development of novel therapeutic approaches.

[Gene expression profiling using oligonucleotide microarray in atrophic gastritis and intestinal metaplasia].

BACKGROUND/AIMS: The atrophic gastritis with intestinal metaplasia of gastric mucosa has been considered to be the major factor of carcinogenesis in the stomach. However, the key molecules are still poorly understood. To elucidate the molecular genetic basis, we report the results of our initial microarray data to analyze the genome pattern in patients with atrophic gastritis and intestinal metaplasia of the stomach. METHODS: We used oligonucleotide microarray technique to evaluate the gene expression profiles in atrophic gastritis with intestinal metaplasia, in comparison with those of normal mucosa. For the identification of differentially expressed genes, Significance Analysis of Microarrays (SAM) package method was used. The results were analyzed using global normalization, intensity dependent normalization, and box plot normalization. RESULTS: Eight genes including FABP, REG, OR6C1, MEP1, SLC6A1, SI, Mucin 1, and RAB23 in mucosa of atrophic gastritis and intestinal metaplasia were up-regulated by more than 10 times as compared with normal gastric mucosa. Only one gene, LOC44119 was down-regulated by more than 10 times of the expression as compared with normal gastric mucosa. In respect to the expression of known genes related to gastric carcinogenesis, 8 genes including FN1, SRMS, TP53, TP53IMP2, TP53I3, FGFR4, TGFB1, and TGFA showed up- and down-regulations more than 2 folds in expression pattern. CONCLUSIONS: We could identify a total genome pattern in patient with atrophic gastritis and intestinal metaplasia using oligonucleotide microarray. We believe that the current results will serve as a fundamental bioinformative basis for clinical applications in diagnosis and treatment of gastric cancer and precancerous lesion in the future.

Profiling of differentially expressed genes in human stem cells by cDNA microarray.

Stem cells are unique cell populations with the ability to undergo both self-renewal and differentiation, although a wide variety of adult stem cells as well as embryonic stem cells have been identified and stem cell plasticity has recently been reported. To identify genes implicated in the control of the stem cell state as well as the characteristics of each stem cell line, we analyzed the expression profiles of genes in human embryonic, hematopoietic (CD34+ and CD133+), and mesenchymal stem cells using cDNA microarrays, and identified genes that were differentially expressed in specific stem cell populations. In particular we were able to identify potential hESC signature-like genes that encode transcription factors (TFAP2C and MYCN), an RNA binding protein (IMP-3), and a functionally uncharacterized protein (MAGEA4). The overlapping sets of 22 up-regulated and 141 down-regulated genes identified in this study of three human stem cell types may also provide insight into the developmental mechanisms common to all human stem cells. Furthermore, our comprehensive analyses of gene expression profiles in various adult stem cells may help to identify the genetic pathways involved in self-renewal as well as in multi-lineage specific differentiation.

Identification and characterization of four novel peptide motifs that recognize distinct regions of the transcription factor CP2.

Although ubiquitously expressed, the transcriptional factor CP2 also exhibits some tissue- or stage-specific activation toward certain genes such as globin in red blood cells and interleukin-4 in T helper cells. Because this specificity may be achieved by interaction with other proteins, we screened a peptide display library and identified four consensus motifs in numerous CP2-binding peptides: HXPR, PHL, ASR and PXHXH. Protein-database searching revealed that RE-1 silencing factor (REST), Yin-Yang1 (YY1) and five other proteins have one or two of these CP2-binding motifs. Glutathione S-transferase pull-down and coimmunoprecipitation assays showed that two HXPR motif-containing proteins REST and YY1 indeed were able to bind CP2. Importantly, this binding to CP2 was almost abolished when a double amino acid substitution was made on the HXPR sequence of REST and YY1 proteins. The suppressing effect of YY1 on CP2's transcriptional activity was lost by this point mutation on the HXPR sequence of YY1 and reduced by an HXPR-containing peptide, further supporting the interaction between CP2 and YY1 via the HXPR sequence. Mapping the sites on CP2 for interaction with the four distinct CP2-binding motifs revealed at least three different regions on CP2. This suggests that CP2 recognizes several distinct binding motifs by virtue of employing different regions, thus being able to interact with and regulate many cellular partners.

Atrophy of brown adipocytes in the adult mouse causes transformation into white adipocyte-like cells.

Adipose tissue is an important endocrine regulator of glucose metabolism and energy homeostasis. Researches have focused on this tissue not only as a target for pharmacotherapy of obesity and insulin resistance but also as an endocrine tissue with leptin secretion and high insulin sensitivity. Brown adipose tissue (BAT) additionally plays a unique role in thermoregulation through the mitochondrial uncoupling protein 1 (UCP1), which uncouples oxidative phosphorylation. As a genetic tissue ablation model of BAT, we made transgenic mice expressing herpes simplex virus thymidine kinase (HSV-TK) driven by the brown adipocyte- specific UCP1 minimal regulatory element. The HSV-TK transgene was expressed specifically in BAT and more than 35% increase of apoptosis was induced by ganciclovir (GCV) treatment. Nevertheless, the expression level was not high enough to induce BAT ablation in GCV-treated adult mice. Importantly, however, we found that brown adipocytes in the periphery of interscapular BAT were transformed into white adipocyte-like unilocular cells. These cells express white adipocyte-specific leptin protein but are different in the ultrastructure of mitochondria from classical white adipocytes. Our data indicates that atrophy of BAT causes transformation into white adipocyte-like cells in the adult mouse and also suggests that further molecular understanding of adipocyte plasticity using our transgenic mouse model might be beneficial for the development of anti-obesity/anti-diabetic therapies.

Purpurogallin exerts anti­inflammatory effects in lipopolysaccharide­stimulated BV2 microglial cells through the inactivation of the NF­κB and MAPK signaling pathways.

In this study, we investigated the inhibitory effects of purpurogallin, a naturally occurring phenol, on the production of lipopolysaccharide (LPS)-induced pro-inflammatory mediators and cytokines in BV2 microglia cells. The cells were pre-treated or not with various concentrations of purpurogallin and then stimulated with 0.5 µg/ml LPS. Cell viability was measured by MTT assay. We also measured the production of nictric oxice (NO) and prostaglandin E2 (PGE2). Our data indicated that treatment with purpurogallin significantly inhibited the excessive production of NO and PGE2 in LPS-stimulated BV2 microglial cells. These inhibitory effects were associated with the downregulation of key enzymes for NO and PGE2, inducible NO synthase (iNOS) and cyclooxygenase-2 (COX2) expression, respectively. Purpurogallin also attenuated the production of pro-inflammatory cytokines, including interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) by suppressing their mRNA and protein expression. The molecular mechanisms underlying the purpurogallin-mediated attenuation of inflammation in BV2 cells closely correlated with the suppression of the translocation of the nuclear factor-κB (NF-κB) p65 subunit into the nucleus and the degradation of the inhibitor of NF-κB (IκB). Moreover, purpurogallin exhibited anti-inflammatory properties by suppressing the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase signaling pathways. These findings suggest that purpurogallin exerts neuroprotective effects through the suppression of pro-inflammatory pathways in activated microglia.

Anti-invasive effects of decitabine, a DNA methyltransferase inhibitor, through tightening of tight junctions and inhibition of matrix metalloproteinase activities in AGS human gastric carcinoma cells.

The DNA methyltransferase inhibitor decitabine, 5-Aza-2'-deoxycytidine, possesses anti-metabolic and anticancer activities in various cancer cells. However, the biochemical mechanisms underlying decitabine-induced inhibition of invasiveness and metastasis have not been thoroughly studied. In this study, we investigated the effect of decitabine on the correlation between tightening of tight junctions (TJs) and anti-invasive activity in AGS human gastric cancer cells. Our data indicated that the inhibitory effects of decitabine on cell motility and invasiveness were associated with increased tightness of the TJ, which was demonstrated by an increase in transepithelial electrical resistance (TER). Immunoblotting results indicated that decitabine repressed the levels of the claudin proteins, major components of TJs that play a key role in the control and selectivity of paracellular transport. Furthermore, matrix metalloproteinase (MMP)-2 and -9 activity in the AGS cells was dose-dependently inhibited by treatment with decitabine, and this was correlated with a decrease in mRNA and protein expression. In addition, these effects were related to inactivation of the phosphoinositide 3-kinase (PI3K)/Akt pathway in AGS cells. In conclusion, this study suggests that TJs and MMPs are critical targets of decitabine-induced inhibition of invasiveness in AGS human gastric cancer cells.

7,8-dihydroxyflavone induces G1 arrest of the cell cycle in U937 human monocytic leukemia cells via induction of the Cdk inhibitor p27 and downregulation of pRB phosphorylation.

We investigated the mechanisms of the anti-proliferative action of 7,8-dihydroxyflavone (7,8-DHF), a member of the flavonoid family, in U937 human monocytic leukemia cells. We found that 7,8-DHF time-dependently inhibited the growth of U937 cells, arresting them in the G1 phase of their cell cycle and inducing apoptosis. 7,8-DHF-induced G1 arrest was correlated with downregulation of cyclin E, with a concomitant upregulation of cyclin-dependent kinase (Cdk) inhibitors including p27, and association of p27 with Cdk2 was markedly induced in 7,8-DHF-treated cells. We also observed that downregulation of the phosphorylation of retinoblastoma protein (pRB) by this flavonoid was associated with enhanced binding of pRB and the transcription factor E2F-1. Overall, our results demonstrate a combined mechanism for the anticancer effects of 7,8-DHF that involves the inhibition of pRB phosphorylation and induction of p27 as targets for 7,8-DHF.

Inhibition of migration and invasion of LNCaP human prostate carcinoma cells by cordycepin through inactivation of Akt.

Cordycepin (3'-deoxyadenosine), a major bioactive compound of Cordyceps militaris, has many pharmacological actions, such as anti-inflammatory and anticancer activities. In this study, the relationship between inhibition of cell motility and anti-invasive activity by cordycepin in LNCaP human prostate carcinoma cells was investigated. Within the concentration range that was not cytotoxic, cordycepin time-dependently inhibited cell motility and invasiveness of LNCaP cells. The inhibitory effects of cordycepin on cell invasiveness were associated with tightening of tight junctions (TJs), which was demonstrated by an increase in transepithelial electrical resistance (TER). Immunoblotting indicated that cordycepin decreases levels of claudin proteins, which are major components of TJs that play a key role in control and selectivity of paracellular transport. Furthermore, cordycepin inhibited the expression and activity of matrix metalloproteinase (MMP)-2 and MMP-9, and simultaneously increased levels of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. These effects were related to inactivation of the phosphoinositide 3-kinase (PI3K)/Akt pathway in LNCaP cells. These findings suggest that cordycepin inhibits the migration and invasion of LNCaP cells by downregulating the activity of TJs and MMPs, possibly in association with suppression of Akt activation.

Immunogenicity of Japanese encephalitis virus envelope protein by Hyphantria cunea nuclear polyhedrosis virus vector in guinea pig.

Japanese encephalitis virus (JEV) is an important pathogen causing febrile syndrome, encephalitis, and death. Envelop (E) glycoprotein is the major target of inducing neutralizing antibodies and protective immunity in host. In this study, E glycoprotein of JEV was expressed in Spodoptera frugiperd 9 cells as a fusion protein containing a gX signal sequence of pseudorabies virus. This purified HcE recombinant protein was evaluated for their immunogenicity and protective efficacy in guinea pig. The survival rates of guinea pig immunized with HcE protein was significantly increased over that of JE vaccine. This result indicates helpful information for developing a subunit vaccine against JEV.

Regulation of NF-kappaB inhibitor IkappaBalpha and viral replication by a KSHV microRNA.

Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to several acquired immune deficiency syndrome-related malignancies, including Kaposi's sarcoma, primary effusion lymphoma (PEL) and a subset of multicentric Castleman's disease. Control of viral lytic replication is essential for KSHV latency, evasion of the host immune system and induction of tumours. Here, we show that deletion of a 14 microRNA (miRNA) cluster from the KSHV genome significantly enhances viral lytic replication as a result of reduced NF-kappaB activity. The miRNA cluster regulates the NF-kappaB pathway by reducing expression of IkappaBalpha protein, an inhibitor of NF-kappaB complexes. Computational and miRNA seed mutagenesis analyses were used to identify KSHV miR-K1, which directly regulates the IkappaBalpha protein level by targeting the 3'UTR of its transcript. Expression of miR-K1 is sufficient to rescue NF-kappaB activity and inhibit viral lytic replication, whereas inhibition of miR-K1 in KSHV-infected PEL cells has the opposite effect. Thus, KSHV encodes an miRNA to control viral replication by activating the NF-kappaB pathway. These results demonstrate an important role for KSHV miRNAs in regulating viral latency and lytic replication by manipulating the host survival pathway.

Low concentrations of doxycycline attenuates FasL-induced apoptosis in HeLa cells

BACKGROUND: Doxycycline (DC) has been shown to possess non-antibiotic properties including Fas/Fas Ligand (FasL)-mediated apoptosis against several tumor types in the concentration range of 10-40 µg/mL. However, the effect of DC in apoptotic signaling at much low concentrations was not studied. METHODS: The present study investigated the attenuation effect of low dose of DC on FasL-induced apoptosis in HeLa cell by the methods of MTT assay, fluorescence microscopy, DNA fragmentation, flow cytometry analysis, and western blotting. RESULTS AND CONCLUSION: In the present findings we showed that low concentration of DC (<2.0 µg/mL) exhibited protective effects against FasL-induced apoptosis in HeLa cells. FasL treatment to HeLa cells resulted in a concentration-dependent induction of cell death, and treatment with low concentrations of DC (0.1-2 µg/mL) significantly (p < 0.001) attenuated the FasL-induced cell death as measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide (MTT) assay. Further, the FasL-induced apoptotic features in HeLa cells, such as morphological changes, DNA fragmentation and cell cycle arrest was also inhibited by DC (0.5 µg/mL). Tetracycline and minocycline also showed similar anti-apoptotic effects but were not significant when compared to DC, tested at same concentrations. Further, DC (0.01-16 µg/mL) did not influence the hydrogen peroxide- or cisplatin-induced intrinsic apoptotic pathway in HeLa cells. Protein analysis using Western blotting confirmed that FasL-induced cleavage/activation of cas-pase-8 and caspase-3, were inhibited by DC treatment at low concentration (0.5 µg/mL). Considering the overall data, we report for the first time that DC exhibited anti-apoptotic effects at low concentrations in HeLa cells by inhibition of caspase activation via FasL-induced extrinsic pathway.

Vertical distraction osteogenesis using a titanium nitride-coated distractor.

OBJECTIVES: The purpose of this study was to examine the effect of using a titanium nitride (TiN)-coated vertical distractor on osseointegration after implantation. STUDY DESIGN: Four adult mongrel dogs, weighing 9-10 kg, were used in this study. The lower premolars were extracted, and vertical distraction was performed after 10 weeks using 8 distraction devices (left, 4 titanium; right, 4 nitrified). A 7-day latency period was allowed before distraction began. The distraction device was activated at a rate of 0.5 mm twice a day for 5 days. After completing distraction, the device was removed after a consolidation period of 6 weeks and 24 implants were installed. The dogs were killed after 4 or 8 weeks. Histologic examinations were performed. RESULTS: The implant success rate was 100% in all of the study groups. Direct bone contact was achieved, and there were no significant differences between the control and experimental groups in the implantation area. CONCLUSION: The results suggested that the nitrified distraction device does not negatively affect osseointegration in the vertical distraction osteogenesis; therefore, it has the advantageous potential to substitute for the conventional distractor.

Chrysanthemum morifolium Ramat (CM) extract protects human neuroblastoma SH-SY5Y cells against MPP+-induced cytotoxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Chrysanthemum morifolium Ramat (Asteraceae) has (CM) long been used in Korean and Chinese traditional herbal medicines with numerous therapeutic applications. AIM OF THE STUDY: To evaluate the neuroprotective activities of Chrysanthemum morifolium (CM) extract against 1-methyl-4-phenylpridinium ions (MPP(+)), Parkinsonian toxin through oxidative stress and impaired energy metabolism, in human SH-SY5Y neuroblastoma cells and the underlying mechanisms. MATERIALS AND METHODS: The effects of CM against MPP(+)-induced cytotoxicity and neuronal cell viability, oxidative damage, the expression of Bcl-2 and Bax, caspase-3 and poly(ADP-ribose) polymerase (PARP) proteolysis were evaluated by using SH-SY5Y neuroblastoma cells. RESULTS: CM effectively inhibited the cytotoxicity and improved cell viability. CM also attenuated the elevation of reactive oxygen species (ROS) level, increase in Bax/Bcl-2 ratio, cleavage of caspase-3 and PARP proteolysis. CONCLUSION: These results demonstrate that CM possesses potent neuroprotective activity and therefore, might be a potential candidate in neurodegenerative diseases such as Parkinson's disease.