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Lipoxygenases (LOXs) are implicated in the biosynthesis of pro- and anti-inflammatory lipid mediators involved in immune cell signaling, most of which catalyze peroxidation of polyunsaturated fatty acids by distinct regio- and stereoselectivity. Current reports suggested that conserved amino acid, Gly in R-LOXs and Ala in S-LOXs, in the catalytic domain play an important role in determining the position as well as the stereochemistry of the functional group. Recently, we have confirmed that the catalytic specificity of cyanobacterial lipoxygenase, named Osc-LOX, with alanine at 296 was 13S-type toward linoleic acid, and producing a 17S- hydroxy-docosahexaenoic acid from docosahexaenoic acid (DHA). Here, we aimed to change the catalytic property of LOX from13S-LOX to 9R-LOX by replacing Ala with Gly and to produce a lipid mediators different from the wild-type using DHA. Finally, we succeeded in generating human endogenous a 13R-hydroxy-docosahexaenoic acid and a 13R,20-dihydroxy-docosahexaenoic acid from DHA through an enzymatic reaction using the Osc-LOX-A296G. Our study could enable physiological studies and pharmaceutical research for the 13R,20-dihydroxy-docosahexaenoic acid.

Omega-3 polyunsaturated fatty acids (PUFAs) have been known to have beneficial effects in the prevention of various diseases. Recently, it was identified that the bioactivities of omega-3 are related to lipid mediators, called pro-resolving lipid mediators (SPMs), converted from PUFAs, so they have attracted much attention as potential pharmaceutical targets. Here, we aimed to build an efficient production system composed of enzymatic and chemical catalysis that converts docosahexaenoic acid (DHA) into lipid mediators. The cyanobacterial lipoxygenase, named Osc-LOX, was identified and characterized, and the binding poses of enzyme and substrates were predicted by ligand docking simulation. DHA was converted into three lipid mediators, a 17S-hydroxy-DHA, a 7S,17S-dihydroxy-DHA (RvD5), and a 7S,15R-dihydroxy-16S,17S-epoxy-DPA (new type), by an enzymatic reaction and deoxygenation. Also, two lipid mediators, 7S,15R,16S,17S-tetrahydroxy-DPA (new type) and 7S,16R,17S-trihydroxy-DHA (RvD2), were generated from 7S,15R-dihydroxy-16S,17S-epoxy-DPA by a chemical reaction. Our study suggests that discovering new enzymes that have not been functionally characterized would be a powerful strategy for producing various lipid mediators. Also, this combination catalysis approach including biological and chemical reactions could be an effective production system for the manufacturing lipid mediators.

Anoctamin1 (ANO1) also known as TMEM16A is a transmembrane protein that functions as a Ca2+ activated chloride channel. Recently, the structure determination of a fungal Nectria haematococca TMEM16 (nhTMEM16) scramblase by X-ray crystallography and a mouse ANO1 by cryo-electron microscopy has provided the insight in molecular architecture underlying phospholipid scrambling and Ca2+ binding. Because the Ca2+ binding motif is embedded inside channel protein according to defined structure, it is still unclear how intracellular Ca2+ moves to its deep binding pocket effectively. Here we show that EF-hand like region containing multiple acidic amino acids at the N-terminus of ANO1 is a putative site regulating the activity of ANO1 by Ca2+ and voltage. The EF-hand like region of ANO1 is highly homologous to the canonical EF hand loop in calmodulin that contains acidic residues in key Ca2+-coordinating positions in the canonical EF hand. Indeed, deletion and Ala-substituted mutation of this region resulted in a significant reduction in the response to Ca2+ and changes in its key biophysical properties evoked by voltage pulses. Furthermore, only ANO1 and ANO2, and not the other TMEM16 isoforms, contain the EF-hand like region and are activated by Ca2+. Moreover, the molecular modeling analysis supports that EF-hand like region could play a key role during Ca2+ transfer. Therefore, these findings suggest that EF-hand like region in ANO1 coordinates with Ca2+ and modulate the activation by Ca2+ and voltage.

Aerobic glycolysis in cancer cells, also known as the Warburg effect, is an indispensable hallmark of cancer. This metabolic adaptation of cancer cells makes them remarkably different from normal cells, thus, inhibiting aerobic glycolysis is an attractive strategy to specifically target tumor cells while sparing normal cells. Macrosphelide A (MSPA), an organic small molecule, is a potential lead compound for the design of anti-cancer drugs. However, its role in modulating cancer metabolism remains poorly understood. MSPA target proteins were screened using mass spectrometry proteomics combined with affinity chromatography. Direct and specific interactions of MSPA with its candidate target proteins were confirmed by in vitro binding assays, competition assays, and simulation modeling. The siRNA-based knockdown of MSPA target proteins indirectly confirmed the cytotoxic effect of MSPA in HepG2 and MCF-7 cancer cells. In addition, we showed that MSPA treatment in the HEPG2 cell line significantly reduced glucose consumption and lactate release. MSPA also inhibited cancer cell proliferation and induced apoptosis by inhibiting critical enzymes involved in the Warburg effect: aldolase A (ALDOA), enolase 1 (ENO1), and fumarate hydratase (FH). Among these enzymes, the purified ENO1 inhibitory potency of MSPA was further confirmed to demonstrate the direct inhibition of enzyme activity to exclude indirect/secondary factors. In summary, MSPA exhibits anti-cancer effects by simultaneously targeting ENO1, ALDOA, and FH.

Brain derived neurotrophic factor (BDNF) promotes maturation of dopaminergic (DAergic) neurons in the midbrain and positively regulates their maintenance and outgrowth. Therefore, understanding the mechanisms regulating the BDNF signaling pathway in DAergic neurons may help discover potential therapeutic strategies for neuropsychological disorders associated with dysregulation of DAergic neurotransmission. Because estrogen‐related receptor gamma (ERRγ) is highly expressed in both the fetal nervous system and adult brains during DAergic neuronal differentiation, and it is involved in regulating the DAergic neuronal phenotype, we asked in this study whether ERRγ ligand regulates BDNF signaling and subsequent DAergic neuronal phenotype. Based on the X-ray crystal structures of the ligand binding domain of ERRγ, we designed and synthesized the ERRγ agonist, (E)-4-hydroxy-N'-(4-(phenylethynyl)benzylidene)benzohydrazide (HPB2) (Kd value, 8.35 μmol/L). HPB2 increased BDNF mRNA and protein levels, and enhanced the expression of the BDNF receptor tropomyosin receptor kinase B (TrkB) in human neuroblastoma SH-SY5Y, differentiated Lund human mesencephalic (LUHMES) cells, and primary ventral mesencephalic (VM) neurons. HPB2-induced upregulation of BDNF was attenuated by GSK5182, an antagonist of ERRγ, and siRNA-mediated ERRγ silencing. HPB2-induced activation of extracellular-signal-regulated kinase (ERK) and phosphorylation of cAMP-response element binding protein (CREB) was responsible for BDNF upregulation in SH-SY5Y cells. HPB2 enhanced the DAergic neuronal phenotype, namely upregulation of tyrosine hydroxylase (TH) and DA transporter (DAT) with neurite outgrowth, both in SH-SY5Y and primary VM neurons, which was interfered by the inhibition of BDNF-TrkB signaling, ERRγ knockdown, or blockade of ERK activation. HPB2 also upregulated BDNF and TH in the striatum and induced neurite elongation in the substantia nigra of mice brain. In conclusion, ERRγ activation regulated BDNF expression and the subsequent DAergic neuronal phenotype in neuronal cells. Our results might provide new insights into the mechanism underlying the regulation of BDNF expression, leading to novel therapeutic strategies for neuropsychological disorders associated with DAergic dysregulation.

Orthodontic treatment involving the bonding of fixed appliances to tooth surfaces can cause white spot lesions (WSLs). WSLs increase the likelihood of cavity formation and hence require preservation and prosthetic restoration. Therefore, the prevention of WSLs is of greater importance than treatment. Application of fluoride or the use of fluoride-containing mouthwash can prevent WSLs, but this requires patient cooperation and additional time and cost. Bioactive glass containing 2.5% fluoride was synthesized and mixed with the orthodontic bonding adhesive Transbond XT Low Flow (LV) at ratios of 1, 3, and 5% to prepare orthodontic adhesive samples. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) were used to characterize the samples. The Vickers hardness test, bracket retention test, and adhesive remnant index (ARI) of the samples were analysed to determine their mechanical properties. To determine the biological cytotoxicity, the cell activity of the samples was evaluated using cell viability tests and the antibacterial activity was analysed using Streptococcus mutans. To evaluate the anti-demineralization effect, the sample was bonded to extracted teeth and a pH cycle test was performed. Micro computed tomography data were obtained from the bonded teeth and sample, and the anti-demineralization effect was evaluated using the ImageJ software program. The Vickers hardness of the sample was higher than that of LV and was dependent on the concentration of fluoride-containing bioactive glass (FBAG). The bracket retention test and ARI of the sample showed no significant differences from those of LV. The cell viability test showed no significant changes at 24 and 48 h after application of the sample. The fluoride ion release test indicated an ion release rate of 9.5&ndash, 17.4 &mu, g/cm2. The antibacterial activity of the experimental group containing FBAG was significantly higher than that of the LV group. The anti-demineralization test showed a concentration-dependent increase. However, the resin containing 5 mass% FBAG (FBAG5) showed a statistically-significant increase compared with LV. The orthodontic adhesive containing FBAG showed antibacterial and anti-demineralization effects, thus indicating possible WSL prevention activity.

All orthodontic appliances are potentially cariogenic. The plaque around the orthodontic appliance can make demineralization on tooth surface causing white spot lesion (WSL). The most effective method to prevent WSL is Fluoride appliance and gargling, but this requires patient cooperation, which consumes additional treatment time and cost. As suggested in this study, biomaterials like bioactive glass and fluorinated graphite (FGt) having antibacterial and anti-demineralization ability effective and easy to use in the clinic. To clinically use orthodontic bonding resins containing Graphite Fluoride BAG (FGtBAG), its properties, biological stability, antimicrobial activity, and remineralization effect must be verified. BAG was mixed with 2.5% FGt containing 51 to 61% fluorine. This mixture was mixed with the CharmFill Flow (CF) in the ratios of 1, 3, and 5 wt%. Microhardness and shear bond strength tests were performed to evaluate its mechanical properties. MTT (3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetra) assay was performed for evaluating its safety. Streptococcus mutans, which is major cariogen by producing lactic acid, was evaluated for antibacterial ability of reducing WSL. In addition, x-ray images were obtained by CBCT (Cone beam computed tomography) after a pH cycle. The remineralization effect was verified in vivo and by Image J. FGtBAG did not differ significantly from CF in mechanical tests. The MTT assay found no significant differences between the groups. The antibacterial activity of FGtBAG at 24 h and 48 h was significantly higher than that of CF. The fluoride release rate tended to increase with the FGtBAG content. The pH cycle results showed that FGtBAG had higher concentration-dependent remineralization effect than CF. The results of this study suggests that orthodontic resins containing FGtBAG can prevent WSL owing to their antibacterial activity and remineralization effect.

The glycoengineering approach is used to improve biophysical properties of protein-based drugs, but its direct impact on binding affinity and kinetic properties for the glycoengineered protein and its binding partner interaction is unclear. Type I interferon (IFN) receptors, composed of IFNAR1 and IFNAR2, have different binding strengths, and sequentially bind to IFN in the dominant direction, leading to activation of signals and induces a variety of biological effects. Here, we evaluated receptor-binding kinetics for each state of binary and ternary complex formation between recombinant human IFN-β-1a and the glycoengineered IFN-β mutein (R27T) using the heterodimeric Fc-fusion technology, and compared biological responses between them. Our results have provided evidence that the additional glycan of R27T, located at the binding interface of IFNAR2, destabilizes the interaction with IFNAR2 via steric hindrance, and simultaneously enhances the interaction with IFNAR1 by restricting the conformational freedom of R27T. Consequentially, altered receptor-binding kinetics of R27T in the ternary complex formation led to a substantial increase in strength and duration of biological responses such as prolonged signal activation and gene expression, contributing to enhanced anti-proliferative activity. In conclusion, our findings reveal N-glycan at residue 25 of R27T is a crucial regulator of receptor-binding kinetics that changes biological activities such as long-lasting activation. Thus, we believe that R27T may be clinically beneficial for patients with multiple sclerosis.

This study aimed to establish maxillary basal arch forms using the root apices and to determine the differences in the basal arch forms in adult women with different sagittal skeletal patterns.This retrospective study included 91 adult women, with either a Class I (n = 24), Class II Division 1 (n = 22), Class II Division 2 (n = 23), or Class III (n = 22) malocclusion, who underwent cone-beam computed tomography. Three-dimensional coordinates of the root apices were determined using the multiplanar reformation mode of OnDemand3D software (Cybermed Inc, Seoul, South Korea). Two-dimensional coordinates were converted from acquired 3-dimensional coordinates via projection on the palatal plane, and the Procrustes superimposition method was used to build the basal arch form. Finally, interroot width measurements were performed for basal arch form comparisons.There were significant differences among the 4 groups (P 0.05) with respect to the intercanine width. The intercanine width of Class II Division 1 group was significantly narrower than that of the other groups. The Class II Division 1 and Class II Division 2 groups tended to have tapered arch forms and squared arch forms, respectively.We established maxillary basal arch forms using the root apices. The Class II Division 1 group had a significantly narrower intercanine distance. The use of the root apex to depict the basal arch form seems reasonable.

White spot lesions (WSL) that occur on teeth after orthodontic appliances have been attached are caused by bacterial demineralization of the enamel surface. This study investigated the anti-demineralization effect of orthodontic resins containing mesoporous bioactive glass nanoparticles (MBN) doped with gallium, which has antibacterial activity, as well as MBN with increased calcium and phosphate contents as these ions can remineralize enamel. Resins (CF, CharmFill Flow, Dentkist, Seoul, South Korea) containing 1%, 3%, and 5% Ga-doped MBN (GaMBN) were characterized using scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and isothermal tests, and their physical properties were measured in terms of Vickers microhardness, bracket retention force, and adhesive remnant index (ARI). Cell viability in the resins was confirmed by testing human dental pulp stem cells (hDPSCs), and ion release tests were performed after 1, 7, and 14 days to determine whether the resins released Ga3+, Ca2+, and PO43&ndash, After 14 days, antibacterial activity was determined using Streptococcus mutans (S. mutans)&mdash, the bacteria that causes tooth decay&mdash, and the chemical remineralization effect was investigated using a cycle of acid&ndash, base solutions. The microhardness of the resins increased with GaMBN concentration whereas their bracket retention force, ARI, and cell viability remained unchanged. The bacterial activity of the 5%-GaMBN resin decreased after 24 and 48 h, however, the change in activity was not statistically significant. Anti-demineralization testing demonstrated that the degree of enamel demineralization decreased as the GaMBN concentration increased, which indicates that resins containing 5%-GaMBN may be viable orthodontic adhesives for preventing WSLs.

The inositol 1,4,5-triphosphate receptor (IP3R), an IP3-gated Ca2+ release channel on the endoplasmic reticulum (ER) membrane, plays a critical role in maintaining cytosolic Ca2+ homeostasis in cells. Particularly, ATP increases IP3R activity by binding to ATPA, a putative glycine-rich Walker A-type motif (GXGXXG) specific to type 1 IP3R (IP3R1). Here, we established an efficient process to produce the ATPA containing domain of IP3R1 (IP3R1-ATPA) using a chaperone co-expression system in Escherichia coli. The recombinant protein was well expressed as a soluble form and showed a high thermostability. Circular dichroism results indicated a mainly α-helical conformation of the purified protein. Additionally, model structures of IP3R1-ATPA were calculated and validated using different modeling algorithms. The structural models of IP3R1-ATPA not only supported the observed high thermostability, but also suggested a potential ATP binding site.

Potent inhibitors of human peptide deformylase (HsPDF) were screened using known PMT analog inhibitors of bacterial peptide deformylase. Forty-three species of PMT analogs that are non-peptidyl bacterial PDF inhibitors like actinonin were selected using virtual screening GOLD. Ten species out of 43 that could bind to HsPDF were selected and their antitumor activities were tested. Among them, four species (PMT-172, PMT-173, PMT-199, and PMT-201) showed excellent growth inhibition of cancer cell in the MTT assay. HsPDF-PMT binding was confirmed through a 1H-CPMG-T2 filter NMR experiment leading to a significant change in peak intensity for PMT-172 and PMT-199. These results suggest that PMT analogs could possibly interact with HsPDF and be a novel anticancer drug candidate.

The latent infection is unique characteristic of Mycobacterium tuberculosis to overcome human immune response for its survival. The M. tb develops adaptation to extreme stress conditions to increase the viability, thus easily acquires drug resistance than any other bacteria and maintains a long-term infection status without any symptoms. Rv0569 is a conserved hypothetical protein that overexpresses under dormant state induced by hypoxia, starvation, and medication. To study function and structure in detail, we determined the solution structure of Rv0569 by NMR. NOE and RDC restraints were used to calculate the structure, which was further refined with AMBER. Rv0569 is composed of five antiparallel β-sheets and one α-helix. Rv0569 shows structural similarity with its homolog Rv2302, yet there is a big difference in the orientation of C-terminal α-helix between Rv0569 and Rv2302. According to previous studies, Rv0569 might comprise a hypoxia induced operon with the Rv0570 which is located 29 bp downstream of the Rv0569 and Rv0570 plays an important role in the latent infection. From our structure and bioinformatics research, we suggest that Rv0569 contributes to signaling transduction in hypoxic condition by binding with DNA for upregulation of Rv0570 or supporting Rv0570 for binding ATP during dormancy of tuberculosis.

We evaluated 3-dimensional changes in the temporomandibular joints of children with skeletal Class III malocclusion and maxillary deficiency after facemask therapy for maxillary protraction. Eighteen children with anterior crossbite and a Class III molar relationship underwent facemask therapy for maxillary protraction, after which they exhibited positive overjet and a Class II molar relationship. Three-dimensional cone-beam computed tomography images of the patients were obtained before (T1) and after (T2) facemask protraction, and the 3-dimensional coordinates of the anatomical landmarks in T1 and T2 images were compared. After facemask therapy, the mandibular condyles of the patients were displaced outside, upward, and backward. Additionally, the anterior and posterior walls of the glenoid fossa had negative values for anteroposterior change. Three-dimensional analysis of the temporomandibular joint showed that facemask therapy resulted in bone apposition (to the anterior wall) and bone resorption (of the posterior wall) in the glenoid fossa. This bone remodeling resulted in upward and backward displacement of the condyle.(J Oral Sci 58, 501-508, 2016).

ARS-Interacting Multi-functional Protein 1 (AIMP1) is a cytokine that is involved in the regulation of angiogenesis, immune activation, and fibroblast proliferation. In this study, fibroblast growth factor receptor 2 (FGFR2) was isolated as a binding partner of AIMP peptide (amino acids 6-46) in affinity purification using human bone marrow-derived mesenchymal stem cells (BMMSCs). AIMP1 peptide induced the proliferation of adult BMMSCs by activating Akt, inhibiting glycogen synthase kinase-3β, and thereby increasing the level of β-catenin. In addition, AIMP1 peptide induced the translocation of β-catenin to the nucleus and increased the transcription of c-myc and cyclin D1 by activating the β-catenin/T-cell factor (TCF) complex. By contrast, transfection of dominant negative TCF abolished the effect of AIMP1. The inhibition of Akt, using LY294002, abolished the accumulation and nuclear translocation of β-catenin induced by AIMP1, leading to a decrease in c-myc and cyclin D1 expression, which decreased the proliferation of BMMSCs. An intraperitoneal injection of AIMP1 peptide into C57/BL6 mice increased the colony formation of fibroblast-like cells. Fluorescence activated cell sorting analysis showed that the colony-forming cells were CD29(+)/CD44(+)/CD90(+)/CD105(+)/CD34(-)/CD45(-), which is characteristic of MSCs. In addition, the fibroblast-like cells differentiated into adipocytes, chondrocytes, and osteocytes. Taken together, these data suggest that AIMP1 peptide promotes the proliferation of BMMSCs by activating the β-catenin/TCF complex via FGFR2-mediated activation of Akt, which leads to an increase in MSCs in peripheral blood.

Objective: In this study, we aimed to examine the relationship between chin deviation and the positional and morphological features of the mandible and to determine the factors that contributed to chin deviation in individuals with a unilateral cleft lip and palate (UCLP). Methods: Cone-beam computed tomography (CBCT) images of 28 adults with UCLP were analyzed in this study. Segmented three-dimensional temporomandibular fossa and mandible images were reconstructed, and angular, linear, and volumetric parameters were measured. Results: For all 28 individuals, the chin was found to deviate to the cleft side by 1.59 mm. Moreover, among these 28 individuals, only 7 showed distinct (more than 4 mm) chin deviation, which was toward the cleft side. Compared to the non-cleft side, the mandibular body length, frontal ramal incli nation, and vertical position of the condyle were lower and inclination of the temporomandibular fossa was steeper on the cleft side. Furthermore, the dif ferences in inclination of the temporomandibular fossa, mandibular body length, ramus length, and condylar volume ratio (non-deviated/deviated) were po sitively correlated with chin deviation. Conclusions: UCLP individuals show mild chin deviation to the cleft side. Statistical differences were noted in the parameters that represented positional and morphological asymmetries of the mandible and temporomandibular fossa; however, these differences were too small to indicate clinical significance. [Korean J Orthod 2013;43(4):168-177]

Immortalization-upregulated protein-1 (IMUP-1) genes have been cloned and are known to be involved in SV40-mediated immortalization. IMUP-1 gene is highly expressed in various cancer cell lines and tumors, suggesting the possibility that they might be involved in tumorigenicity. Previously, there were several problems for overexpression of IMUP-1 in bacterial expression systems including low solubility and aggregation due to unstructured property. To investigate the structural properties, it is necessary to obtain lots of pure and soluble proteins. Accordingly, the co-expression systems of bacterial chaperone proteins, GroEL-GroES, were used to increase solubility of IMUP-1. From the analysis of NMR and CD experiment data, it is suggested that the protein adopt typical the random coil properties in solution.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that negatively regulate gene expression through binding to 3' untranslated region. We identified and characterized the novel miRNA, miR-7641, in human mesenchymal stem cells. The expression of miR-7641 was downregulated during differentiation from human embryonic stem cells to endothelial cells. The CXCL1, a member of the CXC chemokine family, is known as promoting neovascularization by binding G-protein coupled receptors and is related to endothelial cells biogenesis such as angiogenesis, and it was predicted as target gene of miR-7641 by computerized analysis and the luciferase reporter assay. The miR-7641 significantly suppressed CXCL1 of transcriptional and post-translational levels. These data suggest that miR-7641 might be related with differentiation of human endothelial cells.

Objective: The purpose of this study was to investigate the isolation and characterization of multipotent human periodontal ligament (PDL) stem cells and to assess their ability to differentiate into bone, cartilage, and adipose tissue. Methods: PDL stem cells were isolated from 7 extracted human premolar teeth. Human PDL cells were expanded in culture, stained using anti-CD29, -CD34, -CD44, and -STRO-1 antibodies, and sorted by fluorescent activated cell sorting (FACS). Gingival fibroblasts (GFs) served as a positive control. PDL stem cells and GFs were cultured using standard conditions conducive for osteogenic, chondrogenic, or adipogenic differentiation. Results: An average of 152.8 ± 27.6 colony-forming units was present at day 7 in cultures of PDL stem cells. At day 4, PDL stem cells exhibited a significant increase in proliferation (p < 0.05), reaching nearly double the proliferation rate of GFs. About 5.6 ± 4.5% of cells in human PDL tissues were strongly STRO-1-positive. In osteogenic cultures, calcium nodules were observed by day 21 in PDL stem cells, which showed more intense calcium staining than GF cultures. In adipogenic cultures, both cell populations showed positive Oil Red O staining by day 21. Additionally, in chondrogenic cultures, PDL stem cells expressed collagen type II by day 21. Conclusions: The PDL contains multipotent stem cells that have the potential to differentiate into osteoblasts, chondrocytes, and adipocytes. This adult PDL stem cell population can be utilized as potential sources of PDL in tissue engineering applications.

Introduction The purpose of this study was to determine the arch form of the root apices of normally erupting teeth and then determine the differences in the location of the apex of impacted canines relative to normally erupting canines. In addition, we sought to determine whether the labiopalatal position of the impacted canines influences the position of the apices. Methods The study included 21 patients with unerupted canines that subsequently had a normal eruption, 21 patients with palatally impacted canines, 27 patients with labially impacted canines, and 17 patients with midalveolus impacted canines. Images were obtained using cone beam computed tomography, and the x, y, and z coordinates of the root apices were determined using Ondemand3D software (Cybermed Co., Seoul, Korea). Two-dimensional coordinates were converted from acquired 3-dimensional coordinates via projection on a palatal plane, and the Procrustes method was used to process the converted 2-dimensional coordinates and to draw the arch forms of the root apices. Finally, we measured the extent of root apex deviation from the arch forms of the root apices. Results Normally erupting canines showed that even though calcifications may be immature, their positions were aligned with a normal arch form. The root apices of the impacted canines were an average of 6.572 mm away from the root apices' arch form, whereas those of the contralateral nonimpacted canines were an average distance of 2.221 mm away, a statistically significant difference. The palatally impacted canines’ root apices distribution tended toward the first premolar root apices. Conclusions Incompletely calcified, unerupted teeth with a subsequent normal eruption showed a normal arch form of the root apices. The root apices of impacted canines were farther from the arch forms than were the nonimpacted canines. Also, the root apices of impacted canines in the palatal area showed distributions different from those of the other impacted canine groups.

3D structures of STAM1 UIM-ubiquitin complex were presented to predict and analyze the interaction between UIM and ubiquitin. To generate the protein-peptide complex structure, the RosettaDock method was used with and without NMR restraints. High resolution complex structure was acquired successfully and evaluated electrostatic interaction in the protein-peptide binding with several charged residues at the binding site. From docking results, the Rosettadock method could be useful to acquire essential information of protein-protein or protein-peptide interaction with minimal biological evidences.

HP1298 (Swiss-Prot ID ; P65108) is an 72-residue protein from Helicobacter pylori strain 26695. The function of HP1298 was identified as Translation initiation factor IF-l based on sequence homology, and HP1298 is included in IF-l family. Here, we report the sequence-specific backbone resonance assignments of HP1298. About 97% of all the , , , , and resonances could be assigned unambiguously. We could predict the secondary structure of HP1298, by analyzing the deviation of the and shemical shifts from their respective random coil values. Secondary structure prediction shows that HP1298 consists of six -strands. This study is a prerequisite for determining the solution structure of HP1298 and investigating the structure-function relationship of HP1298. Assigned chemical shift can be used for the study on interaction between HP1298 and other Helicobacter pylori proteins.

The aim of this article was to determine whether mechanical forces combined with LLLT stimulate expression of OPG, RANKL, and RANK in animal model by immunohistochemistry. A total of 20 sprague-dawley rats at 15 weeks old were divided into two groups of 10 rats each, to form the laser irradiation and nonirradiation groups. Total of 20 mg of orthodontic force was applied to rat incisors. The duration of tooth movement was 1, 3, 7, 14, and 21 days. A Ga-Al-As diode laser was used to irradiate the area around the moved teeth for 10 seconds once a day for 7 days. Proliferative activities of OPG, RANKL, and RANK were evaluated immunohistochemically. After the application of force, the induction of OPG, RANKL, and RANK was observed on both groups. In the laser irradiation group, however, the expression of OPG, RANKL, and RANK was significantly greater than that of the nonirradiation group at day 1 to day 21. This is the first study about immunohistological findings of RANK/RANKL/OPG triad under LLLT during tooth movement. These findings suggest that low level laser therapy can stimulate bone turnover during experimental orthodontic tooth movement.

Matrix metalloproteinases (MMPs) play important roles in normal brain development and synaptic plasticity, although aberrant expression of MMPs leads to brain damage, including blood–brain barrier disruption, inflammation, demyelination, and neuronal cell death. In this article, we report that MMP-8 is upregulated in LPS-stimulated BV2 microglial cells and primary cultured microglia, and treatment of MMP-8 inhibitor (M8I) or MMP-8 short hairpin RNA suppresses proinflammatory molecules, particularly TNF-α secretion. Subsequent experiments showed that MMP-8 exhibits TNF-α–converting enzyme (TACE) activity by cleaving the prodomain of TNF-α (A74/Q75, A76/V77 residues) and, furthermore, that M8I inhibits TACE activity more efficiently than TAPI-0, a general TACE inhibitor. Biochemical analysis of the underlying anti-inflammatory mechanisms of M8I revealed that it inhibits MAPK phosphorylation, NF-κB/AP-1 activity, and reactive oxygen species production. Further support for the proinflammatory role of microglial MMP-8 was obtained from an in vivo animal model of neuroinflammatory disorder. MMP-8 is upregulated in septic conditions, particularly in microglia. Administration of M8I or MMP-8 short hairpin RNA significantly inhibits microglial activation and expression/secretion of TNF-α in brain tissue, serum, and cerebrospinal fluid of LPS-induced septic mice. These results demonstrate that MMP-8 critically mediates microglial activation by modulating TNF-α activity, which may explain neuroinflammation in septic mouse brain.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase (RdRp), is the key enzyme for HCV RNA replication. We previously showed that HCV RdRp is phosphorylated by protein kinase C-related kinase 2 (PRK2). In the present study, we used biochemical and reverse-genetics approaches to demonstrate that HCV NS5B phosphorylation is crucial for viral RNA replication in cell culture. Two-dimensional phosphoamino acid analysis revealed that PRK2 phosphorylates NS5B exclusively at its serine residues in vitro and in vivo . Using in vitro kinase assays and mass spectrometry, we identified two phosphorylation sites, Ser29 and Ser42, in the Δ1 finger loop region that interacts with the thumb subdomain of NS5B. Colony-forming assays using drug-selectable HCV subgenomic RNA replicons revealed that preventing phosphorylation by Ala substitution at either Ser29 or Ser42 impairs HCV RNA replication. Furthermore, reverse-genetics studies using HCV infectious clones encoding phosphorylation-defective NS5B confirmed the crucial role of these PRK2 phosphorylation sites in viral RNA replication. Molecular-modeling studies predicted that the phosphorylation of NS5B stabilizes the interactions between its Δ1 loop and thumb subdomain, which are required for the formation of the closed conformation of NS5B known to be important for de novo RNA synthesis. Collectively, our results provide evidence that HCV NS5B phosphorylation has a positive regulatory role in HCV RNA replication. IMPORTANCE While the role of RNA-dependent RNA polymerases (RdRps) in viral RNA replication is clear, little is known about their functional regulation by phosphorylation. In this study, we addressed several important questions about the function and structure of phosphorylated hepatitis C virus (HCV) nonstructural protein 5B (NS5B). Reverse-genetics studies with HCV replicons encoding phosphorylation-defective NS5B mutants and analysis of their RdRp activities revealed previously unidentified NS5B protein features related to HCV replication and NS5B phosphorylation. These attributes most likely reflect potential structural changes induced by phosphorylation in the Δ1 finger loop region of NS5B with two identified phosphate acceptor sites, Ser29 and Ser42, which may transiently affect the closed conformation of NS5B. Elucidating the effects of dynamic changes in NS5B phosphorylation status during viral replication and their impacts on RNA synthesis will improve our understanding of the molecular mechanisms of NS5B phosphorylation-mediated regulation of HCV replication.

The bacterial toxin-antitoxin (TA) system is a module that may play a role in cell survival under stress conditions. Generally, toxin molecules act as negative regulators in cell survival and antitoxin molecules as positive regulators. Thus, the expression levels and interactions between toxins and antitoxins should be systematically harmonized so that bacteria can escape such harmful conditions. Since TA systems are able to control the fate of bacteria, they are considered potent targets for the development of new antimicrobial agents. TA systems are widely prevalent with a variety of systems existing in bacteria: there are three types of bacterial TA systems depending on the property of the antitoxin which binds either the protein toxin or mRNA coding the toxin protein. Moreover, the multiplicity of TA genes has been observed even in species of bacteria. Therefore, knowledge on TA systems such as the individual characteristics of TA systems, integrative working mechanisms of various TA systems in bacteria, interactions between toxin molecules and cellular targets, and so on is currently limited due to their complexity. In this regard, it would be helpful to know the structural characteristics of TA modules for understanding TA systems in bacteria. Until now, 85 out of the total structures deposited in PDB have been bacterial TA system proteins including TA complexes or isolated toxins/antitoxins. Here, we summarized the structural information of TA systems and analyzed the structural characteristics of known TA modules from several bacteria, especially focusing on the TA modules of several infectious bacteria.

Helicobacter pylori (H. pylori) have a unique ability to survive in extreme acidic environments and to colonize the gastric mucosa. It can cause diverse gastric diseases such as peptic ulcers, chronic gastritis, mucosa-associated lymphoid tissue (MALT) lymphoma, gastric cancer, etc. Based on genomic research of H. pylori, over 1600 genes have been functionally identified so far. However, H. pylori possess some genes that are uncharacterized since: (i) the gene sequences are quite new; (ii) the function of genes have not been characterized in any other bacterial systems; and (iii) sometimes, the protein that is classified into a known protein based on the sequence homology shows some functional ambiguity, which raises questions about the function of the protein produced in H. pylori. Thus, there are still a lot of genes to be biologically or biochemically characterized to understand the whole picture of gene functions in the bacteria. In this regard, knowledge on the 3D structure of a protein, especially unknown or hypothetical protein, is frequently useful to elucidate the structure-function relationship of the uncharacterized gene product. That is, a structural comparison with known proteins provides valuable information to help predict the cellular functions of hypothetical proteins. Here, we show the 3D structures of some hypothetical proteins determined by NMR spectroscopy and X-ray crystallography as a part of the structural genomics of H. pylori. In addition, we show some successful approaches of elucidating the function of unknown proteins based on their structural information.

STAM1 and Hrs are the components of ESCRT-0 complex for lysosomal degradation of membrane proteins is composed of STAM1 Hrs and has multiple ubiquitin binding domains. Here, the solution structure of STAM1 UIM, one of the ubiquitin binding motif, was determined by NMR spectroscopy. The structure of UIM adopts an α-helix with amphipathic nature. The central hydrophobic residues in UIM provides the binding surface for ubiquitin binding and are flanked with positively and negatively charged residues on both sides. The docking model of STAM1 UIM-ubiquitin complex is suggested. In NMR and ITC experiments with the specifically designed mutant proteins, we investigated the ubiquitin interaction of tandem ubiquitin binding domains from STAM1. The ubiquitin binding affinity of the VHS domain and UIM in STAM1 was 52.4 and 94.9 μM, and 1.5 and 2.2 fold increased, respectively, than the value obtained from the isolated domain or peptide. The binding affinities here would be more physiologically relevant and provide more precise understanding in ESCRT pathway of lysosomal degradation.

Granular glands in the skins of frogs synthesize and secrete a remarkably diverse range of peptides capable of antimicrobial activity. These anuran skin antimicrobial peptides are commonly hydrophobic, cationic and form an amphipathic α-helix in a membrane mimetic solution. Recently, they have been considered as useful target molecules for developing new antibiotics drugs. Esculentin-1c is a 46-amino acid residue peptide isolated from skin secretions of the European frog, Rana esculenta. It displays the most potent antimicrobial activity among bioactive molecules. Esculentin-1c has the longest amino acids among all antimicrobial peptides. The present study solved the solution structure of esculentin-1c in TFE/water by NMR, for the first time. We conclude that this peptide is comprised of three α-helices with each helix showing amphipathic characteristics, which seems to be a key part for permeating into bacterial membranes, thus presenting antimicrobial activity.

The aim of this study was to investigate the effects of low-level laser (LLL) irradiation on the turnover of fibronectin and collagen type I in periodontal tissue during tooth movement in rats by immunohistochemistry. Thirty male Sprague-Dawley rats aged 15 weeks were assigned to either an experimental group (n = 15) that underwent LLL irradiation during tooth movement, or a control group (n = 15). In the experimental group, the gallium-aluminum-arsenide (Ga-Al-As) diode LLL (wavelength 808 nm; output 96 mW) was used to irradiate three areas on both the palatal side and the labial side of the maxillary incisor. The radiation was administered by the contact method for 10 s at 0.83 J/cm(2) energy dose, once a day for 7 days. Total energy dose over the complete schedule was 34.86 J/cm(2). The animals were killed on days 1, 3, 7, 14 and 21. There was no difference between the two groups in the amount of tooth movement. The immunohistochemistry results showed that the expression of fibronectin and collagen type I in the experimental group had significantly increased from day 1, with a more even distribution than in the control group, and that this difference was maintained until the end of the experiment. These results suggest that LLL irradiation facilitates the reorganization of the connective tissues during tooth movement in rats.

Acyl carrier protein (ACP) is a small acidic protein and its primary structure is highly conserved in various bacterial sources. Despite its small size, it interacts with diverse proteins associated with many biosynthetic pathways. The three-dimensional structure of H. pylori ACP and its structural characteristics were clarified using NMR and CD spectroscopy. H. pylori ACP consists of four helices connected by different sized loops. The helices correspond to residues L3-Q14 (alphaI), S36-G50 (alphaII), D56-E60 (alphaIII), and V65-K76 (alphaVI). The size of each helix differs slightly from that of homologous ACPs. However, H. pylori ACP showed a distinct pH-dependent conformational characteristic: at neutral pH, it adopts a partially unfolded structure, while it has a tight fold at pH 6. The chemical shift perturbation and (1)H-(15)N steady state NOE analysis at both pH 6 and 7 showed that the local change of structural components occurred mainly around loop II, and this change was reflected by the changes of the residues Ile 54 and Asp 56. Examination of the structure showed that the network of Glu 47, Ile 54, Asn 75, and Lys 76 is very important for the structural stability. The pH-dependent folding process shows a kind of cooperativity, since all the residues involved in the conformational transitions are contiguous and in spatial proximity.

One of the small proteins from Helicobacter pylori, acyl carrier protein (ACP), was investigated by NMR. ACP is related to various cellular processes, especially with the biosynthesis of fatty acid. The basic NMR resonance assignment is a prerequisite for the validation of a heterologous protein interaction with ACP in H. pylori. Here, the results of the backbone (1)H, (15)N, and (13)C resonance assignments of the H. pylori ACP are reported using double- and triple-resonance techniques. About 97% of all of the (1)HN, (15)N, (13)CO, (13)Calpha, and (13)Cbeta resonances that cover 76 of the 78 non-proline residues are clarified through sequential- and specific- assignments. In addition, four helical regions were clearly identified on the basis of the resonance assignments.

At pH6 added filamentous bacteriophage fd is compatible with many of the detergents used to solubilize membrane proteins for solution NMR studies of membrane proteins and, therefore, serves as an alignment media. In combination with strained polyacrylamide gel alignment, Dipolar Waves can be used to directly assess the secondary structure and a lambda-map extracts the order tensors for de novo structure calculation of membrane proteins without distance restraints.