Antioxidative effects of ethyl 2-(3-(benzo[d]thiazol-2-yl)ureido)acetate against amyloid ß-induced oxidative cell death via NF-κB, GSK-3ß and ß-catenin signaling pathways in cultured cortical neurons.

We have previously shown that 2-(3-(benzo[d]thiazol-2-yl)ureido)acetate (KHG21834) attenuates amyloid beta(Aß)25-35-induced apoptotic death and shows anti-inflammatory activity against Aß25-35-induced microglial activation. However, antioxidative effects of KHG21834 against Aß-induced oxidative stress have not yet been reported. In the present study, we investigated the antioxidative function of KHG21834 in primary cultured cortical neurons, to expand the potential therapeutic efficacy of KHG21834. Pretreatment with KHG21834 protected against Aß-induced neuronal cell death and mitochondrial damage, and significantly restored GSH levels and the activities of catalase, superoxide dismutase, and glutathione peroxidase, and also suppressed the production of reactive oxygen species and protein oxidation. These results imply that KHG21834 may play a role in cellular defense mechanisms against Aß-induced oxidative stress in cultured cortical neurons. Furthermore, KHG21834 significantly attenuated the effects of Aß treatment on levels of NF-κB, ß-catenin, and GSK-3ß proteins in cortical neurons. Taken together, our results suggest that the antioxidant effects of KHG21834 may result at least in part from its ability to regulate the NF-κB, ß-catenin, and GSK-3ß signaling pathways. To our knowledge, this is the first report showing that KHG21834 significantly attenuates Aß25-35-induced oxidative stress in primary cortical neurons, and provides novel insights into KHG21834 as a possible therapeutic agent for the treatment of Aß-mediated neurotoxicity involving oxidative stress.

P42 Ebp1 regulates the proteasomal degradation of the p85 regulatory subunit of PI3K by recruiting a chaperone-E3 ligase complex HSP70/CHIP.

The short isoform of ErbB3-binding protein 1 (Ebp1), p42, is considered to be a potent tumor suppressor in a number of human cancers, although the mechanism by which it exerts this tumor-suppressive activity is unclear. Here, we report that p42 interacts with the cSH2 domain of the p85 subunit of phosphathidyl inositol 3-kinase (PI3K), leading to inhibition of its lipid kinase activity. Importantly, we found that p42 induces protein degradation of the p85 subunit and further identified HSP70/CHIP complex as a novel E3 ligase for p85 that is responsible for p85 ubiquitination and degradation. In this process, p42 couples p85 to the HSP70/CHIP-mediated ubiquitin-proteasomal system (UPS), thereby promoting a reduction of p85 levels both in vitro and in vivo. Thus, the tumor-suppressing effects of p42 in cancer cells are driven by negative regulation of the p85 subunit of PI3K.

Computer-navigated total knee arthroplasty in haemophilic arthropathy.

Total knee arthroplasty (TKA) in end-stage haemophilic arthropathy is complex and challenging due to the altered bony anatomy, arthrofibrosis and muscle contractures. Computer navigation is especially advocated in patients with deformity or altered anatomy to improve alignment and to assist in ligament balancing. The objective of this study was to evaluate the results of computer-navigated TKA in haemophilic arthropathy. A consecutive series of computer-assisted TKA for the end-stage haemophilic arthropathy between February 2007 and December 2009 were evaluated. A total of 27 TKA were performed in 25 patients. Pre- and postoperative full-length weight-bearing radiographs were assessed for the axial limb alignment. The orientation of the components was measured on anteroposterior radiographs. Clinically, Knee Society score and Short Form-36 were evaluated. The mechanical axis of the leg was within a range of ±3° varus/valgus in 92% of the TKA. The coronal alignment of the femoral and tibial components was within a range of ±3 degrees in 96% of the knees. The clinical outcomes were significantly improved after the operation. There were no complications specific to the computer navigation. Computer-navigated TKA helps in restoring the mechanical axis and improves accuracy of orientation of the components in patients with end-stage haemophilic arthropathy. Potential benefits in long-term outcome require further investigation.

Injection-molded nanocomposites and materials based on wheat gluten.

This is, to our knowledge, the first study of the injection molding of materials where wheat gluten (WG) is the main component. In addition to a plasticizer (glycerol), 5 wt.% natural montmorillonite clay was added. X-ray indicated intercalated clay and transmission electron microscopy indicated locally good clay platelet dispersion. Prior to feeding into the injection molder, the material was first compression molded into plates and pelletized. The filling of the circular mold via the central gate was characterized by a divergent flow yielding, in general, a stronger and stiffer material in the circumferential direction. It was observed that 20-30 wt.% glycerol yielded the best combination of processability and mechanical properties. The clay yielded improved processability, plate homogeneity and tensile stiffness. IR spectroscopy and protein solubility indicated that the injection molding process yielded a highly aggregated structure. The overall conclusion was that injection molding is a very promising method for producing WG objects.

Gene delivery to human adult and embryonic cell-derived stem cells using biodegradable nanoparticulate polymeric vectors.

Gene delivery to stem cells holds great potential for tissue regeneration and delivery of therapeutic proteins. The major barrier is the lack of safe and efficient delivery methods. Here, we report enhanced gene delivery systems for human stem cells using biodegradable polymeric vectors. A library of poly (beta-amino esters) end-modified derivatives was developed and optimized for high transfection efficiency and low cytotoxicity for three human stem cell lines including human mesenchymal stem cells (hMSCs), human adipose-derived stem cells (hADSCs) and human embryonic stem cell-derived cells (hESCds). In the presence of 10% serum, leading end-modified C32 polymeric vectors exhibited significantly high transfection efficiency in hMSCs (27+/-2%), hADSCs (24+/-3%) and hESCds (56+/-11%), with high cell viability (87-97%) achieved in all cell types. Our results show that poly(beta-amino esters) as a class, and end-modified versions of C32 in particular, are efficient polymeric vectors for gene delivery to both adult and embryonic-derived stem cells.

Growth effects of botulinum toxin type A injected into masseter muscle on a developing rat mandible.

OBJECTIVES: Botulinum toxin type A (BTX-A) reduces the muscular contractions by temporarily inhibiting the release of acetylcholine at the neuromuscular junction. The purpose of this study was to investigate the effects of the BTX-A injected into the masseter muscle of a developing rat mandible. MATERIALS AND METHODS: Four-week-old male (no. 80) Sprague-Dawley rats were divided into four groups: control group, saline group, BTX-A group and baseline control group. Rats of baseline group were sacrificed at 0 day to provide baseline values of the mandibular measurements. The masseter muscle of rats in the saline and the BTX-A group were administered with saline and BTX-A solutions respectively. Experimental animals were sacrificed after 4 weeks. RESULTS: The BTX-A group demonstrated smaller mandibular dimension compared with the other groups (P < 0.05). Their condylar cartilages showed increased apoptosis at the proliferation stage of the reserve zone and masseter muscle fibers demonstrated atrophic changes. CONCLUSIONS: The result demonstrated BTX-A influence on inhibitory action of the developing mandible because of apoptosis at the proliferation stage of the reserve zone of the condylar cartilage in developing rat mandible.

The effect of cortical activation on orthodontic tooth movement.

OBJECTIVE: Cortical activation is one of the procedures to accelerate tooth movement by manipulating the cortical bone. In this study, the effect of cortical activation on orthodontic tooth movement was investigated clinically and histologically in the surrounding bony tissue. MATERIALS AND METHODS: In the lower and upper jaws of two beagle dogs, cortical activation was applied to the buccal and lingual side of the alveolar bone in the right jaw where 12 holes were made on each cortical plate 4 weeks after the extraction of all the second bicuspids while under deep anesthesia. All third bicuspids on both jaws were forced to move forward by a 150-g force using NiTi coil spring with/without guiding wire. The tooth movement was measured and the animals were killed after tooth movement. RESULTS: Rapid initial tooth movement was apparent after cortical activation. However, after 6 months of cortical activation, the cell number and cellular activity of the surrounding periodontal tissue were decreased. CONCLUSIONS: This experiment showed that rapid initial tooth movement was apparent following the application of orthodontic force after cortical activation but the cellular activity and fibroblast structure were abnormal in the surrounding periodontal tissue.

Alteration of BMP-4 and Runx2 expression patterns in mouse temporomandibular joint after ovariectomy.

OBJECTIVE: Temporomandibular disorder (TMD) includes a number of clinical conditions involving the masticatory musculature or the temporomandibular joint (TMJ) and associated structures. Previous studies have shown the presence of high-affinity estrogen receptors in the TMJ articular cartilage. The aim of this study was to evaluate the developmental changes in mouse TMJ under estrogen deficiency. MATERIALS AND METHODS: Four-month-old ovariectomized mice were killed after certain weeks. We examined the significant alterations of the expression patterns of bone morphogenetic protein (BMP)-4, Runx2, and bone sialoprotein (BSP) after ovariectomy. RESULTS: In the control group, BMP-4, Runx2, and BSP expressions showed no definite difference at any stage. In the ovariectomy group, the intensity of BMP-4 and Runx2 expression increased after ovariectomy. BSP immunoreactivity, however, increased slightly at 2 weeks but then decreased gradually. CONCLUSIONS: Estrogen plays important roles in the metabolism and maintenance of TMJ via regulations of signaling molecules such as BMP-4, Runx2, and BSP. Our results suggest that estrogen deficiency is a candidate cause of TMD. This study revealed further osteogenetic properties of estrogen that may be useful in the clinical treatment and prevention of TMD.

Inhibition of apoptosis in early tooth development alters tooth shape and size.

Apoptosis plays important roles in various stages of organogenesis. In this study, we hypothesized that apoptosis would play an important role in tooth morphogenesis. We examined the role of apoptosis in early tooth development by using a caspase inhibitor, z-VAD-fmk, concomitant with in vitro organ culture and tooth germ transplantation into the kidney capsule. Inhibition of apoptosis at the early cap stage did not disrupt the cell proliferation level when compared with controls. However, the macroscopic morphology of mice molar teeth exhibited dramatic alterations after the inhibition of apoptosis. Crown height was reduced, and mesiodistal diameter was increased in a concentration-dependent manner with z-VAD-fmk treatment. Overall, apoptosis in the enamel knot would be necessary for the proper formation of molar teeth, including appropriate shape and size.

The time course of biological and immunochemical allergy states induced by anisakis simplex larvae in rats.

Oral infection by Anisakis simplex third stage larvae (L3) frequently gives rise to an allergic response. To comprehend the allergic and immune responses induced by L3, we investigated the kinetics of specific antibody isotype expression and the time course of biological and immunochemical allergy states using sera prepared from rats orally infected with L3 twice, with an interval of 9 weeks between infections. Biological and immunochemical allergy states were analysed by RBL-2H3 exocytosis and by indirect ELISA for IgE, respectively. The peak IgM at reinfection (RI) was comparable or similar to that at primary infection (PI) both in levels analysed by indirect ELISA and in antigen recognition analysed by Western blot. IgG1 and IgG2a levels were higher and showed accelerated kinetics after RI vs. after PI. However, the level of IgG2b was substantially lower than that of IgG2a. Peak immunochemical and biological allergy states for RI were higher and were reached faster than those for PI. The peak biological allergy state was observed at 1 week postreinfection and this occurred sooner than that for the peak immunochemical allergy state found at 2 weeks postreinfection. Our analysis of the relationship between specific IgE avidity and biological allergy state did not show any meaningful correlation. These results suggest that the allergic response induced by L3 oral infection is predominantly caused by reinfection and that this is accompanied by an elevated IgM level, which further suggests that the biological allergy state might not be related to specific IgE avidity.

Developmental properties of the Hertwig's epithelial root sheath in mice.

Hertwig's epithelial root sheath (HERS) plays an important role in tooth root formation. In this study, we examined root formation of the first molar in mice, focusing on cell proliferation, cell death, cell migration, and the expression patterns of the signaling molecules, including glycoproteins and proteoglycans between PN8 and PN26. The number of HERS cells decreased during root formation, although HERS retained total length until PN15. The migration of HERS cells did not occur during root formation. Moreover, the immunopositive reaction of laminin beta-3 and syndecan-1 in HERS indicates that both cell adhesion and cell proliferation are essential for HERS development. Bmp-2, Bmp-4, and Msx-2 were expressed in HERS cells during root formation. We also developed an in vitro culture system for investigating the periodontium and suggest that this system provides an excellent vehicle for full exploration, and hence improved understanding, of the development and regeneration of the periodontium. Together, our results provide a comprehensive model describing the morphogenesis of early root development in vertebrates.

Inactivation of human glutamate dehydrogenase by aluminum.

Aluminum inactivated glutamate dehydrogenase (GDH) by a pseudo-first-order reaction at micromolar concentrations. A double-reciprocal plot gave a straight line with a k(inact) of 2.7 min(-1) and indicated the presence of a binding step prior to inactivation. The inactivation was strictly pH dependent and a marked increase in sensitivity to aluminum was observed as the pH decreased. At a pH higher than 8.5, no inactivation was observed. The completely inactivated GDH contained 2 mol of aluminum per mole of enzyme subunit monomer. When preincubated with enzyme, several chelators such as citrate, NaF, N-(2-hydroxyethyl) ethylenediaminetriacetic acid or ethylenediaminetriacetic acid efficiently protected the enzyme against the aluminum inactivation. In a related experiment, only citrate and NaF released the aluminum from the completely inactivated aluminum-enzyme complex and fully recovered the enzyme activity. Ferritin, NADP+, or nerve growth factor did not show any effects on the recovery of the aluminum-inactivated GDH activity. The dissociation constant for the aluminum-enzyme complex was calculated to be 5.3 microM. Although aluminum has been known to form a complex with nucleotides, no such effects were observed in the inactivation of GDH by aluminum as determined using GDHs mutated at the ADP-binding site, NAD+-binding site or GTP-binding site. Circular dichroism studies showed that the binding of aluminum to the enzyme induced a decrease in alpha helices and beta sheets and an increase in random coil. Therefore, inactivation of GDH by aluminum is suggested to be due to the conformational change induced by aluminum binding. These results suggest a possibility that aluminum-induced alterations in enzymes of the glutamate system may be one of the causes of aluminum-induced neurotoxicity.