dickkopf-3-related gene regulates the expression of zebrafish myf5 gene through phosphorylated p38a-dependent Smad4 activity.

Myf5 is a myogenic regulatory factor that functions in myogenesis. An intronic microRNA, miR-In300, located within zebrafish myf5 intron I, has been reported to silence myf5 through the targeting of dickkopf-3-related gene (dkk3r). However, the molecular mechanism underlying the control of myf5 expression by dkk3r is unknown. By injecting dkk3r-specific morpholino-oligonucleotide (dkk3r-MO) to knock down Dkk3r, we found that the phosphorylated p38a protein was reduced. Knockdown of p38a resulted in malformed somites and reduced myf5 transcripts, which photocopied the defects induced by injection of dkk3r-MO. To block the MAPK pathway, phosphorylation of p38 was inhibited by introduction of SB203580, which caused the down-regulation of myf5 expression. The GFP signal was dramatically decreased in somites when we injected p38a-MO into embryos derived from transgenic line Tg(myf5(80K):GFP), in which the GFP was driven by the myf5 promoter. Although these p38a-MO-induced defects were rescued by co-injection with p38a mRNA, they were not rescued with p38a mRNA containing a mutation at the phosphorylation domain. Moreover, overexpression of Smad2 or Smad3a enhanced myf5 expression, but the defects induced by the dominant negative form of either Smad2 or Smad3a equaled those of embryos injected with either dkk3r-MO or p38a-MO. These results support the involvement of Smad2·Smad3a in p38a mediation. Overexpression of Smad4 enabled the rescue of myf5 defects in the dkk3r-MO-injected embryos, but knockdown of either dkk3r or p38a caused Smad4 protein to lose stability. Therefore, we concluded that Dkk3r regulates p38a phosphorylation to maintain Smad4 stability, in turn enabling the Smad2·Smad3a·Smad4 complex to form and activate the myf5 promoter.

Novel intronic microRNA represses zebrafish myf5 promoter activity through silencing dickkopf-3 gene.

A strong, negative cis-element located at the first intron +502/+835 (I300) of zebrafish myf5 has been reported. To elucidate the molecular mechanism underlying this repression network, we microinjected zebrafish single-cell embryos with I300 RNA, resulting in the dramatic reduction of luciferase activity driven by the myf5 promoter. Within this I300 segment, we identified an intronic microRNA (miR-In300) located at +609/+632 and found that it was more highly expressed in the older mature somites than those newly formed, which negatively correlated with the distribution of zebrafish myf5 transcripts. We proved that miR-In300 suppressed the transcription of myf5 through abolishing myf5 promoter activity, and we subsequently identified the long isoform of the Dickkopf-3 gene (dkk3) as the target gene of miR-In300. We further found that injection of the dkk3-morpholinos (MOs) resulted in downregulation of myf5 transcripts in somites, whereas co-injection of myf5 mRNA with dkk3-MO1 enabled rescue of the defects induced by dkk3-MO1 alone. Finally, injection of miR-In300-MO enhanced both myf5 transcripts in somites and the level of Dkk3 protein in zebrafish embryos. Based on these findings, we concluded that miR-In300 binds to its target gene dkk3, which inhibits the translation of dkk3 mRNA and, in turn, suppresses zebrafish myf5 promoter activity.

Altered prostate epithelial development and IGF-1 signal in mice lacking the androgen receptor in stromal smooth muscle cells.

BACKGROUND: Androgens and the androgen receptor (AR) play critical roles in the prostate development via mesenchymal-epithelial interactions. Smooth muscle cells (SMC), differentiated from mesenchyme, are one of the basic components of the prostate stroma. However, the roles of smooth muscle AR in prostate development are still obscure. METHODS: We established the smooth muscle selective AR knockout (SM-ARKO) mouse model using the Cre-loxP system, and confirmed the ARKO efficiency at RNA, DNA and protein levels. Then, we observed the prostate morphology changes, and determined the epithelial proliferation, apoptosis, and differentiation. We also knocked down the AR in a prostate smooth muscle cell line (PS-1) to confirm the in vivo findings and to probe the mechanism. RESULTS: The AR was selectively and efficiently knocked out in the anterior prostates of SM-ARKO mouse. The SM-ARKO prostates have defects with loss of infolding structures, and decrease of epithelial proliferation, but with little change of apoptosis and differentiation. The mechanism studies showed that IGF-1 expression level decreased in the SM-ARKO prostates and AR-knockdown PS-1 cells. The decreased IGF-1 expression might contribute to the defective development of SM-ARKO prostates. CONCLUSIONS: The AR in SMCs plays important roles in the prostate development via the regulation of IGF-1 signal.

A novel DNA selection and direct extraction process and its application in DNA recombination.

In the conventional bench-top approach, the DNA recombination process is time- and effort-consuming due to laborious procedures lasting from several hours to a day. A novel DNA selection and direct extraction process has been proposed, integrated and tested on chip. The integrative microfluidic chip can perform the whole procedure of DNA recombination, including DNA digestion, gel electrophoresis, DNA extraction and insert-vector ligation within 1 h. In this high-throughput design, the manual gel cutting was replaced by an automatic processing system that performed high-quality and high-recovery efficiency in DNA extraction process. With no need of gel-dissolving reagents and manipulation, the application of selection and direct extraction process could significantly eliminate the risks from UV and EtBr and also facilitate DNA recombination. Reliable output with high success rate of cloning has been achieved with a significant reduction in operational hazards, required materials, efforts and time.

Transforming growth factor beta2 regulates growth and differentiation of pulp cells via ALK5/Smad2/3.

Transforming growth factor beta (TGF-beta) may regulate the biological activities of dental pulp cells. We found that human dental pulp cells expressed TGF-beta1, TGF-beta2, and a little amount of TGF-beta3 messenger RNA (mRNA). The exposure of pulp cells to TGF-beta2 induced the phosphorylation of Smad2/3, Smad1/5/8, and extracellular regulated-kinase 1/2 (ERK1/2) as observed by Western blotting. Exposure to TGF-beta2 decreased the alkaline phosphatase (ALP) mRNA expression and enzyme activity. Pretreatment of pulp cells with SB431542 (an inhibitor of TGF-beta ALK-4, ALK-5, and ALK-7 receptors) but not U0126 (a MEK1 inhibitor) prevented the inhibition of viable cell number, ALP activity, and mRNA expression by TGF-beta2 in dental pulp cells. These results suggest that TGF-beta may affect the growth and differentiation of dental pulp cells via an autocrine fashion by activation of the ALK/Smad2/3-signal transduction pathways. TGF-beta2 possibly regulates the differentiation of pulp cell at specific stages synergistically with other factors.

A novel tetraspanin C189 upregulated in C6/36 mosquito cells following dengue 2 virus infection.

Dengue (Den) viruses cause apoptosis in mammalian cells, but usually result in high progeny yields without evident damage in mosquito cells. By using subtractive hybridization, 13 potentially virus-induced genes were selected in Den-2 virus-infected Aedes albopictus C6/36 cells. Based on semi-quantitative and real-time RT-PCR, one novel gene, named C189, was significantly upregulated in infected C6/36 cells. Its full-length of 678 nucleotides (nt) was determined by a combination of 5'- and 3'-RACE products. After alignment, C189 was classified as a member of the tetraspanin superfamily that typically has 2 short cytoplasmic sequences, 4 transmembrane domains, as well as small and large extracellular regions (EC1 and EC2). It contains the hallmark CCG motif in the EC2 region and additional 17 conserved nucleotides as do other tetraspanins. C189 was not upregulated by inoculation of UV-inactivated Den-2 virus to C6/36 cells. This suggests that tetraspanin upregulation is not related to virus binding to the cell surface, and that C189 does not function as a receptor for dengue virus entry. On the other hand, overexpression of C189 was concurrent with viral proteins, targeting the plasma membrane of C6/36 cells infected with Den-2 virus. It is presumably beneficial or essential for cell-to-cell spread of the virus due to the role of tetraspanins demonstrated in intercellular adhesion.

Alpha-vitamin E derivative, RRR-alpha-tocopheryloxybutyric acid inhibits the proliferation of prostate cancer cells.

AIM: To investigate the activity of RRR-alpha-tocopheryloxybutyric acid (TOB), an ether analog of RRR-alpha-tocopheryl succinate (VES), in prostate cancer cells. METHODS: VES and TOB were used to treat prostate cancer LNCaP, PC3, and 22Rv1 cells and primary-cultured prostate fibroblasts. The proliferation rates were determined by MTT assay, the cell viabilities were determined by trypan blue exclusion assay, and the cell deaths were evaluated by using Cell Death Detection ELISA kit. The protein expression levels were determined by Western blot analysis. RESULTS: The MTT growth assay demonstrated that TOB could effectively suppress the proliferation of prostate cancer cells, but not normal prostate fibroblasts. Mechanism dissections revealed that TOB reduced cell viability and induced apoptosis in prostate cancer cells similar to VES. In addition, both TOB and VES suppressed prostate-specific antigen (PSA) at the transcriptional level leading to reduced PSA protein expression. Furthermore, vitamin D receptor (VDR) expression increased after the addition of TOB. CONCLUSION: Our data suggests that the VES derivative, TOB, is effective in inhibiting prostate cancer cell proliferation, suggesting that TOB could be used for both chemopreventive and chemotherapeutic purposes in the future.

Parallel infection of Japanese encephalitis virus and Wolbachia within cells of mosquito salivary glands.

The endosymbiont Wolbachia usually causes cytoplasmic incompatibility in dipteran hosts, including mosquitoes. However, some important arbovirus-transmitting mosquitoes such as Aedes aegypti (L.) are not heritably infected by Wolbachia. In Wolbachia-harboring mosquito Armigeres subalbatus Coquillett, colocalization of Wolbachia and inoculated Japanese encephalitis virus (family Flaviviridae, genus Flavivirus, JEV) in salivary gland (SG) cells was shown by electron microscopy. The infection rate of JEV in SGs, detected with either immunofluorescent antibody test or reverse transcription-polymerase chain reaction, did not show significant differences between Wolbachia-infected and -free colonies. It is suggested that Wolbachia did not mediate resistance of SG cells to superinfection by JEV, although both microorgamisms coexist in the same niche, i.e., the same SG cell. Therefore, a SG escape barrier may not be elevated due to Wolbachia infection, which presumably has no deleterious effects on vector competence in Wolbachia-harboring mosquitoes.

Antiplatelet effect by p-cresol, a uremic and environmental toxicant, is related to inhibition of reactive oxygen species, ERK/p38 signaling and thromboxane A2 production.

P-cresol is a well-known uremic toxin and environmental toxicant that may affect platelet functions. In this study, p-cresol (1-5 µM) inhibited the arachidonic acid (AA)-induced platelet aggregation, with 47% and 82% of inhibition at concentrations of 2 and 5 µM, respectively. Under similar experimental condition, p-cresol showed little effect on the U46619-induced platelet aggregation. p-cresol (<500 µM) revealed no discernable cytotoxicity to platelets as analyzed by quantification of lactate dehydrogenase release. Antiplatelet effect of p-cresol was related to inhibition of thromboxane A(2) (TXA(2)) and prostaglandin D(2) (PGD(2)) formation. P-cresol (2-100 µM) partly inhibited the AA-induced reactive oxygen species (ROS) production as well as the extracellular signal-regulated kinase (ERK1/2) and p38 phosphorylation in platelets. P-cresol further inhibited the AA-induced aggregation of rabbit platelet-rich plasma (PRP) with an IC50 of 2 µM and aggregation of human PRP (IC50 = 13.6 µM). Intravenous administration of p-cresol (250-1000 nmole) into mice effectively suppressed the ex vivo platelet aggregation, whereas showed little effect on the value of RBC, hemoglobin (HGB), hematocrit, MCV, MCH, MCHC, platelets and lymphocyte counts. These results indicate that in acute p-cresol-poisoning and long-term exposure to cresol as in severe uremic patients, p-cresol may potentially inhibit blood clot formation and lead to hemorrhagic disorders via inhibition of platelet aggregation, ROS production, ERK/p38 activation and TXA(2) production.

Cemental tear: clinical characteristics and its predisposing factors.

INTRODUCTION: Cemental tears often show characteristics mimicking a periapical or periodontal lesion. This leads to difficulty in the early diagnosis of cemental tears. METHODS: In this multicenter study, 71 teeth with cemental tears being confirmed by direct inspection or histological examination were included. For each case, demographic data, dental history, clinical and radiographic findings, and the results of exploratory surgery were recorded and analyzed. RESULTS: Maxillary or mandibular incisors (76.1%) were most frequently affected by cemental tears. Univariate analysis of predisposing factors found that teeth with cemental tears occurred more commonly in men (77.5%) and patients older than 60 years of age (73.2%). Analysis of clinical characteristics showed that teeth with cemental tears were prone to have abscess formation (66.2%), a deep pocket >6 mm (73.2%), positive vitality test (65.3%), healthy antagonist teeth (84.3%), and moderate to severe attrition (77.9%). About 56.3% of cemental tears could be detected on preoperative radiographs. Further analysis of radiographic findings showed that teeth with cemental tears were more likely to have periodontal bone destruction (85.9%) or periapical bone destruction (64.8%). CONCLUSIONS: Endodontists and dentists may avoid misdiagnosis and unnecessary treatment of teeth with cemental tears if they can properly evaluate the radiographs and pulp vitality of teeth as well as know the predisposing factors and clinical characteristics of teeth with cemental tears in advance.

Areca nut-induced micronuclei and cytokinesis failure in Chinese hamster ovary cells is related to reactive oxygen species production and actin filament deregulation.

Epidemiological studies have shown a strong association between environmental exposure to betel quid (BQ) and oral cancer. Areca nut (AN), an ingredient of BQ, contains genotoxic and mutagenic compounds. In this study, we found that AN extract (ANE) inhibited the growth of Chinese hamster ovary cells (CHO-K1) in a dose- and time-dependent manner. Intracellular reactive oxygen species (ROS) levels and micronuclei (MN) frequency were significantly increased following ANE treatment in CHO-K1 cells. Addition of catalase markedly inhibited ANE-induced MN formation, indicating that ANE-induced genotoxicity was correlated with intracellular H(2)O(2). Incubation of CHO-K1 cells with ANE (400-800 microg/ml) for 24 hr caused G2/M arrest, and prolonged exposure to ANE (800 microg/ml) significantly induced cell death. Surprisingly, ANE itself caused cytokinesis failure and subsequent increase in binucleated cell formation. Coexposure to catalase (2,000 U/ml) and ANE (800 microg/ml) reduced the generation of binucleated cells, indicating that ANE-induced cytokinesis failure was associated with oxidative stress. Following prolonged exposure to ANE, an accumulation of hyperploid/aneuploid cells concomitant with bi-, micro- or multinucleated cells was found. In summary, our results demonstrate that ANE exposure to CHO-K1 cells caused increased MN frequency, G2/M arrest, cytokinesis failure, and an accumulation of hyperploid/aneuploid cells. These events are associated with an increase in intracellular H(2)O(2) level and actin filament disorganization.

Prostaglandin F(2alpha)-induced interleukin-8 production in human dental pulp cells is associated with MEK/ERK signaling.

Prostaglandin F(2alpha) (PGF(2alpha)) and interleukin-1beta (IL-1beta) levels are elevated in inflamed dental pulp. The roles of IL-1beta and PGF(2alpha) in the pathogenesis of pulpal inflammation await investigation. We found that IL-1beta stimulated PGF(2alpha) production of human dental pulp cells. IL-1beta and PGF(2alpha) (0.5-10 mumol/L) also induced IL-8 production and mRNA expression in pulp cells. Aspirin inhibited IL-1beta-induced PGF(2alpha), but not IL-8 production. PGF(2alpha)-induced IL-8 production and mRNA expression were inhibited by U0126 (an inhibitor of mitogen-activated protein kinase kinase [MEK1/2]) inhibitor), whereas SQ22536 (an adenylate cyclase inhibitor) enhanced this event. These results indicate that IL-1beta-induced IL-8 production in pulp cells is not mainly via direct activation of cyclooxygenase and PGF(2alpha) generation. PGF(2alpha)-induced IL-8 production is possibly via activation of MEK/extracellular signal-regulated kinase signaling, but not by activation of adenylate cyclase. IL-1beta and PGF(2alpha) might involve the pathogenesis of pulpal inflammation via induction of IL-8 production.

The effect of BisGMA on cyclooxygenase-2 expression, PGE2 production and cytotoxicity via reactive oxygen species- and MEK/ERK-dependent and -independent pathways.

After operative restoration, some monomers released from dentin bonding agents or composite resin may induce tissue inflammation and affect the vitality of dental pulp. Whether BisGMA, a major monomer of composite resin, may induce prostaglandin release and cytotoxicity to pulp cells and their mechanisms awaits investigation. We found that BisGMA induced cytotoxicity to human dental pulp cells at concentrations higher than 0.075 mm as analyzed by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. BisGMA (0.1 mm) also stimulated ERK phosphorylation, PGE(2) production, COX-2 mRNA and protein expression as well as ROS production (as indicated by an increase in cellular DCF fluorescence) in dental pulp cells. Catalase (500 and 1000 U/ml) and U0126 (10 and 20 microm, a MEK inhibitor) effectively prevented the BisGMA-induced ERK activation, PGE(2) production and COX-2 expression. Moreover, catalase can protect the pulp cells from BisGMA cytotoxicity, whereas aspirin and U0126 lacked of this protective activity. These results suggest that BisGMA released from composite resin may potentially affect the vitality of dental pulp and induce pulpal inflammation via stimulation of ROS production, MEK/ERK1/2 activation and subsequent COX-2 gene expression and PGE(2) production. Cytotoxicity of BisGMA to dental pulp cells is related to ROS production, but not directly mediated by MEK activation and PGE(2) production.

Comparative cytotoxicity of five current dentin bonding agents: role of cell cycle deregulation.

To compare the cytotoxicity of three nano-dentin bonding agents (nano-DBAs) and two non-nano-DBAs using Chinese hamster ovary (CHO-K1) cells. We found that nano fillers were not the major contributing factor in DBA cytotoxicity, as analyzed by colony forming assay and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Exposure of CHO-K1 cells to all three tested total-etching DBAs led to G(0)/G(1) cell cycle arrest, whereas exposure to higher concentrations of two tested nano-DBAs induced G(2)/M arrest. All five DBAs further induced apoptosis at the highest concentration, as analyzed by propidium iodide staining flow cytometry. The toxicity of all DBAs (1:4000v/v or higher) is related to increased reactive oxygen species (ROS) production, as analyzed by single cell DCF fluorescence flow cytometry. These results indicate that clinical application of DBAs may be potentially toxic to dental pulp tissues. Cytotoxicity of DBAs is associated with ROS production, cell cycle deregulation and apoptosis. Presence of methacrylate monomers such as PENTA and UDMA is possibly the major cytotoxic factor for DBAs. Further studies on other toxicological endpoints of nano-DBAs are necessary to highlight their safe use.

Persistent endodontic lesion due to complex cementodentinal tears in a maxillary central incisor--a case report.

The cementodentinal tear is rarely detected by noninvasive procedures owing to its clinical picture simulating a root fracture or a periodontal or endodontic lesion. We present a case of complex cementodentinal tears in a 79-year-old woman who presented a repeated swelling at the labial mucosa of the left maxillary central incisor for 6 months. Periapical radiographs demonstrated a vertical radiolucent fracture line extending from the root apex along the mesial aspect of the root to near the middle portion of the root of the left maxillary central incisor. Because endodontic re-treatment failed to cure the disease, periapical surgery was performed, and 2 fractured U-shaped root fragments around the apical root surface were removed. Histologic examination showed that the 2 fractured root fragments were composed mainly of the dentin covered by a thin layer of the cementum and overlying periodontal ligament tissue, suggesting cementodentinal tears. A swelling recurred 8 months after the initial operation. Therefore, a second periapical surgery was performed. Although no obvious fracture line was observed around the root surface, the second surgery did not cure the disease, either. A persistent small swelling was noted at the alveolar mucosa of the affected tooth during the follow-up. We conclude that although a cementodentinal tear can be detected by a careful radiographic examination, its clinical outcome is not predictable by surgical removal only.