Curcumin Derivative GT863 Inhibits Amyloid-Beta Production via Inhibition of Protein N-Glycosylation.

Amyloid-ß (Aß) peptides play a crucial role in the pathogenesis of Alzheimer's disease (AD). Aß production, aggregation, and clearance are thought to be important therapeutic targets for AD. Curcumin has been known to have an anti-amyloidogenic effect on AD. In the present study, we performed screening analysis using a curcumin derivative library with the aim of finding derivatives effective in suppressing Aß production with improved bioavailability of curcumin using CHO cells that stably express human amyloid-ß precursor protein and using human neuroblastoma SH-SY5Y cells. We found that the curcumin derivative GT863/PE859, which has been shown to have an inhibitory effect on Aß and tau aggregation in vivo, was more effective than curcumin itself in reducing Aß secretion. We further found that GT863 inhibited neither ß- nor γ-secretase activity, but did suppress γ-secretase-mediated cleavage in a substrate-dependent manner. We further found that GT863 suppressed N-linked glycosylation, including that of the γ-secretase subunit nicastrin. We also found that mannosidase inhibitors that block the mannose trimming step of N-glycosylation suppressed Aß production in a similar fashion, as was observed as a result of treatment with GT863. Collectively, these results suggest that GT863 downregulates N-glycosylation, resulting in suppression of Aß production without affecting secretase activity.

Inhibition of microtubule assembly competent tubulin synthesis leads to accumulation of phosphorylated tau in neuronal cell bodies.

Neurofibrillary tangles, a pathological hallmark of Alzheimer's disease (AD), are somatodendritic filamentous inclusions composed of hyperphosphorylated tau. Microtubule loss is also a common feature of affected neurons in AD. However, whether and how the disruptions of microtubules and the microtubule-associated proteins occur in the pathogenesis of AD remain unclear. Recent evidence indicates that reduced expression of tubulin by knocking down a tubulin chaperon can cause tau neurotoxicity. Thus, the disruption of tubulin homeostasis may result in the acquisition of tau pathogenesis and ultimately cause tauopathy. To investigate whether the disruption of tubulin maintenance induces tau abnormalities in mammalian neurons, we developed a miRNA-mediated knockdown system of tubulin-specific chaperon E (Tbce), which is a factor required for the de novo synthesis of tubulin. Tbce knockdown in mouse primary cultured neurons induced an increase in tubulin in the cell body at 14 days in vitro. Accumulated tubulin was not acetylated or incorporated in microtubules, indicating that they were functionally inert. Concomitantly, tau also accumulated in neuronal cell bodies. The mis-localized tau was phosphorylated at Ser202/Thr205 and Ser396/Ser404. These results indicate that Tbce knockdown in mammalian neurons induces not only a reduction in properly folded tubulins, which are microtubule assembly competent, but also an accumulation of phosphorylated tau in the cell body of mammalian neurons. These findings suggest that disruption of the homeostatic mechanism for maintaining tubulin biosynthesis and/or microtubules can cause tau accumulation in the cell body, which is commonly observed in tauopathies.

Associations between serum 25-hydroxyvitamin D3 level and skeletal muscle mass and lower limb muscle strength in Japanese middle-aged subjects.

OBJECTIVES: One of the important risk factors of falling is decreased muscle mass and muscle strength. Recently, there has been an increasing concern on the role of vitamin D in muscle strength and physical activity. Aim of our study is to examine the relationships between vitamin D status and muscle mass and muscle strength in middle-aged healthy adults. METHODS: Subjects were 40 healthy volunteers aged 42.0 ± 10.6 years old. Evaluation was made for serum vitamin D3 metabolites including 25-hydroxyvitamin D3 [25(OH)D3] and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] concentrations, lower limb muscle strength, and dietary intake by food frequency questionnaire. Body composition was measured by dual-energy X-ray absorptiometry (DXA), and appendicular skeletal mass index (ASMI) was calculated as skeletal muscle mass/squared height. RESULTS: 70% of the subjects had vitamin D insufficiency/deficiency (serum total 25(OH)D < 20 ng/mL), and female subjects had significantly lower serum total 25(OH)D level compared with males. Vitamin D insufficiency/deficiency group had significantly higher body fat, lower SMI and muscle strength, probably reflecting higher percentage of female subjects. Serum vitamin D3 metabolites levels were significantly correlated with whole and site-specific ASMI, and lower limb muscle strength, except for the correlation between serum 24,25(OH)2D3 concentration and lower limb muscle strength. In addition, serum 25(OH)D3 level was a positive significant predictor for both ASMI and lower limb muscle strength, while serum 24,25(OH)2D3 level was not their significant predictor. CONCLUSIONS: Serum 25(OH)D3 level was significantly correlated with both skeletal muscle mass and lower limb muscle strength.

The A673T mutation in the amyloid precursor protein reduces the production of ß-amyloid protein from its ß-carboxyl terminal fragment in cells.

INTRODUCTION: The A673T mutation in the amyloid precursor protein (APP) protects against Alzheimer's disease by reducing ß-amyloid protein (Aß) production. This mutation reduced the release of the soluble APP fragment (sAPPß), which is processed by ß-secretase, suggesting a concomitant decrease in the ß-carboxyl fragment of APP (C99), which is a direct substrate of γ-secretase for Aß production. However, it remains controversial whether the level of C99 is significantly reduced in cells expressing APP that carry A673T as the cause of reduced Aß production. Here, we investigated the effect of the A673T mutation in C99 on γ-cleavage in cells. RESULTS: We found that the level of C99 in cells expressing APP A673T was indistinctive of that observed in cells expressing wild-type APP, although the release of sAPPß was significantly reduced in the APP A673T cells. In addition, our reconstituted ß-secretase assay demonstrated no significant difference in ß-cleavage on an APP fragment carrying the A673T mutation compared with the wild-type fragment. Importantly, cells expressing C99 containing the A673T mutation (C99 A2T; in accordance with the Aß numbering) produced roughly half the level of Aß compared with the wild-type C99, suggesting that the C99 A2T is an insufficient substrate of γ-secretase in cells. A cell-free γ-secretase assay revealed that Aß production from the microsomal fraction of cells expressing C99 A2T was diminished. A sucrose gradient centrifugation analysis indicated that the levels of the C99 A2T that was codistributed with γ-secretase components in the raft fractions were reduced significantly. CONCLUSIONS: Our data indicate that the A673T mutation in APP alters the release of sAPPß, but not the C99 level, and that the C99 A2T is an inefficient substrate for γ-secretase in cell-based assay.

γ-Secretase associated with lipid rafts: multiple interactive pathways in the stepwise processing of ß-carboxyl-terminal fragment.

γ-Secretase generates amyloid ß-protein (Aß), a pathogenic molecule in Alzheimer disease, through the intramembrane cleavage of the ß-carboxyl-terminal fragment (ßCTF) of ß-amyloid precursor protein. We previously showed the framework of the γ-secretase cleavage, i.e. the stepwise successive processing of ßCTF at every three (or four) amino acids. However, the membrane integrity of γ-secretase was not taken into consideration because of the use of the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized reconstituted γ-secretase system. Here, we sought to address how the membrane-integrated γ-secretase cleaves ßCTF by using γ-secretase associated with lipid rafts. Quantitative analyses using liquid chromatography-tandem mass spectrometry of the ßCTF transmembrane domain-derived peptides released along with Aß generation revealed that the raft-associated γ-secretase cleaves ßCTF in a stepwise sequential manner, but novel penta- and hexapeptides as well as tri- and tetrapeptides are released. The cropping of these peptides links the two major tripeptide-cleaving pathways generating Aß40 and Aß42 at several points, implying that there are multiple interactive pathways for the stepwise cleavages of ßCTF. It should be noted that Aß38 and Aß43 are generated through three routes, and γ-secretase modulator 1 enhances all the three routes generating Aß38, which results in decreases in Aß42 and Aß43 and an increase in Aß38. These observations indicate that multiple interactive pathways for stepwise successive processing by γ-secretase define the species and quantity of Aß produced.

Orbicularis oris myomucosal island flap transfer to the nose.

We developed the orbicularis oris myomucosal island flap (OOMMIF) to reconstruct the nasal lining in one stage. The OOMMIF blood supply derives from the intramuscular vascular network which communicates with the submucosal vascular plexus via the vascular network formed by the deep ascending branches of the superior labial artery. An oral mucosal flap of approximately 2 x 3cm can be harvested from the upper lip pedicled solely on the orbicularis oris muscle. We transferred this flap to a nasal lining defect located in the ala in four patients, the nasal floor in two patients, and the columella in two patients. The flap donor site was closed primarily. All flaps took completely with satisfactory results. Minor complications included slight asymmetry of the vermilion height due to donor site contracture in one patient and flap drooping in two patients corrected by secondary debulking. Upper lip functional loss was not observed, although upper lip hypoaesthesia occurred in one patient, which disappeared within 6 months. An OOMMIF can be easily elevated with minimal donor site morbidity. Thus, the OOMMIF is a good candidate for one-stage reconstruction of small nasal lining defects.

Efficacy of distraction osteogenesis for mandibular reconstruction in previously irradiated areas: clinical experiences.

The efficacy of distraction osteogenesis in an irradiated area is controversial, although this procedure is now widely used in the field of craniomaxillofacial surgery. We report the clinical results from 4 patients with mandibular defects treated by lengthening of the irradiated mandibles. All patients had a mandibular defect caused by ablation of a malignant tumor. They had undergone radiotherapy at a total dose of 30 to 50 Gy to the surgical site after tumorectomy. Distraction osteogenesis was used as the secondary reconstruction method in 6 sites of the remaining irradiated mandibles and in 1 site of the transferred vascularized scapula after radiotherapy. The transported segment was obtained by corticotomy with an initial gap of 0 to 2 mm, and internal extension plates were used. Distraction was commenced after a latency period of 7 to 10 days and performed at the rate of 0.25 to 1.0 mm/d. The total amount of distraction and consolidation periods ranged from 15 to 25 mm and 120 to 193 days, respectively. In 5 of the 6 sites in the remaining irradiated mandibles, satisfactory bone formation in the distraction gap was observed, although a fracture after new bone formation was observed in 1 site. Fibrous callus formation was observed in 1 irradiated site only, and satisfactory results were obtained in another site of transferred vascularized scapula in the same patient. From these experiences, we believe that distraction may provide a reconstruction option for mandibular defects even under irradiated conditions because the procedure is simple and less invasive.

Reconstruction of a severe maxillofacial deformity after tumorectomy and irradiation using distraction osteogenesis and LeFort I osteotomy before vascularized bone graft.

We present the successful reconstruction of a large mandibular defect with a severe maxillofacial deformity after malignant tumor resection and irradiation. The patient was a 16-year-old boy with a defect in the left mandible, which extended from the mandibular body to the condylar process and hypoplasia of the maxillozygomatic complex on the left side as a result of ablation and radiotherapy of a grown rhabdomyosarcoma in the left infratemporal fossa at the age of 10. We planned a two-stage reconstruction because of his wide mandibular defect and hypoplasia. LeFort I type osteotomy to correct the maxillary declination was combined with mandibular lengthening to decrease the width of the defect in the first stage. New bone formation was confirmed at the distraction site 4 months after surgery, and the second stage was performed. A free latissimus dorsi myocutaneous flap with a vascularized scapula and rib was transferred to reconstruct the ramus of the mandible, zygomatic arch, and soft tissues. This procedure resulted in satisfactory results. In conclusion, the combination of distraction osteogenesis and microsurgical bone transplantation facilitated the straightforward reconstruction of a three-dimensional deformity with huge bony defects. We think that this combined surgical procedure will become a favorable option in the treatment of severe maxillomandibular deformities with bone defects.

Distinguishing between proliferating nodal lymphoid blasts in chronic myelogenous leukemia and non-Hodgkin lymphoma: report of three cases and detection of a bcr/abl fusion signal by single-cell analysis.

Lymph node biopsies were analyzed from three patients with chronic myelogenous leukemia (CML) showing nodal blast proliferation. Immunohistochemically, the blasts from all three patients had an immature marker profile with a T-blast population (cCD3+, CD4-, CD7+, CD8-, CD99+, terminal deoxynucleotidyl transferase +) and a hematopoietic progenitor cell marker (CD34). In two patients, the blasts also expressed myeloid lineage specificity (naphthol AS-D chloroacetate esterase activity and myeloperoxidase positivity). However, it was difficult to distinguish between blast proliferation in CML and non-Hodgkin lymphoma from these immunohistopathological findings alone. Subsequently, bcr gene rearrangement and bcr/abl mRNA expression were detected by Southern blot and reverse transcription-polymerase chain reaction analysis of the lymph nodes. Fluorescence in situ hybridization (FISH) analysis of lymph node touch smears also disclosed bcr/abl gene fusion signals in the blasts of all patients, confirming that the blasts were derived from Philadelphia chromosome-positive CML. Accurate discrimination between the proliferating nodal blasts of CML and non-Hodgkin lymphoma is essential for determining subsequent therapy. FISH analysis of bcr/abl in single-cell blast preparations is an efficient tool that allows rapid, accurate cytopathological diagnosis of extramedullary blast-phase CML and its discrimination from non-Hodgkin lymphoma.