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Rationale : Neuroinflammation is a primary feature of Alzheimer's disease (AD), for which an increasing number of drugs have been specifically developed. The present study aimed to define the therapeutic impact of a specific subpopulation of T cells that can suppress excessive inflammation in various immune and inflammatory disorders, namely, CD4 + CD25 + Foxp3 + regulatory T cells (Tregs). Methods : To generate Aβ antigen-specific Tregs (Aβ + Tregs), Aβ 1-42 peptide was applied in vivo and subsequent in vitro splenocyte culture. After isolating Tregs by magnetic bead based purification method, Aβ + Tregs were adoptively transferred into 3xTg-AD mice via tail vein injection. Therapeutic efficacy was confirmed with behavior test, Western blot, quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry staining (IHC). In vitro suppression assay was performed to evaluate the suppressive activity of Aβ + Tregs using flow cytometry. Thy1.1 + Treg trafficking and distribution was analyzed to explore the infused Tregs migration into specific organs in an antigen-driven manner in AD mice. We further assessed cerebral glucose metabolism using 18 F-FDG-PET, an imaging approach for AD biological definition. Subsequently, we evaluated the migration of Aβ + Tregs toward Aβ activated microglia using live cell imaging, chemotaxis, antibody blocking and migration assay. Results : We showed that Aβ-stimulated Tregs inhibited microglial proinflammatory activity and modulated the microglial phenotype via bystander suppression. Single adoptive transfer of Aβ + Tregs was enough to induce amelioration of cognitive impairments, Aβ accumulation, hyper-phosphorylation of tau, and neuroinflammation during AD pathology. Moreover, Aβ-specific Tregs effectively inhibited inflammation in primary microglia induced by Aβ exposure. It may indicate bystander suppression in which Aβ-specific Tregs promote immune tolerance by secreting cytokines to modulate immune responses during neurodegeneration. Conclusions : The administration of Aβ antigen-specific regulatory T cells may represent a new cellular therapeutic strategy for AD that acts by modulating the inflammatory status in AD., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Particulate matter (PM), a component of air pollution, has been epidemiologically associated with a variety of diseases. Recent reports reveal that PM has detrimental effects on the brain. In this study, we aimed to investigate the biological effects of ambient particles on the neurodegenerative disease Parkinson's disease (PD). We exposed mice to coarse particles (PM 10 : 2.5-10 μm) for short (5 days) and long (8 weeks) durations via intratracheal instillation. Long-term PM 10 exposure exacerbated motor impairment and dopaminergic neuron death in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse models. Short-term PM 10 exposure resulted in both pulmonary and systemic inflammatory responses in mice. We further investigated the mechanism underlying PM 10 -induced neurotoxicity in cocultures of lung LA-4 epithelial cells and RAW264.7 macrophages. PM 10 treatment elicited a dramatic increase in proinflammatory mediators in LA-4/RAW264.7 coculture. Treating BV2 microglial cells with PM 10 -treated conditioned medium induced microglial activation. Furthermore, 1-methyl-4-phenylpyridinium (MPP + ) treatment caused notable cell death in N2A neurons cocultured with activated BV2 cells in PM 10 -conditioned medium. Altogether, our results demonstrated that PM 10 plays a role in the neurodegeneration associated with PD. Thus, the impact of PM 10 on neurodegeneration could be related to detrimental air pollution-induced systemic effects on the brain.

Measuring displacement is essential for assessing the safety of bridges. Non-contact sensors such as vision sensors can precisely measure displacement but may be expensive or incapable of micro-scale measurement at a low cost, unlike contact displacement sensors, which are economical but challenging to install. This study proposes an economical, remote non-contact sensor system. The system comprises a laser beam transmitter and a light receiver, deriving the displacement based on the position where the laser beam is irradiated to the light-receiving surface. To measure this, the light receiver was installed at the measurement point and included a wireless communicator to transmit the displacement data. A displacement experiment was conducted to evaluate the performance. The results confirmed that precise displacement measurements were possible at a resolution of 100 µm. For bridge load tests, a light receiver under a bridge was installed, laser beams irradiated to the light-receiving surface from a distance, and the displacement was measured for each test and compared with the values measured by a conventional contact sensor. The results were highly consistent with those of the existing sensor, indicating that the proposed sensor system applies to bridge loading tests and the safety diagnosis for various structures.

Cardiac myxoma typically is thought to be a slow-growing, benign primary. Atrial myxomas can lead to many complications and can also mimic mitral stenosis, infective endocarditis, and other vascular diseases associated with systemic embolization. A 75-year-old woman with a history of lung cancer (pT1cN1, adenocarcinoma), atrial fibrillation, and a cerebral infarction presented with dysarthria and visual disturbances. In our case, we had to consider some questionable issues with the left atrial mass, and whether the recurrence of cerebral events was due to the thrombotic material in the left atrium or from locally recurrent lung cancer from the stump margin of the previously resected left superior pulmonary vein. We present a case with a rapidly-growing left atrial myxoma with a growth rate of 12.60 mm/month, rather than a thrombus or local recurrence of tumor under a medication of non-VKA oral antagonists.

Parkinson's disease (PD) is a chronic progressive neurodegenerative movement disorder characterized by the selective loss of dopaminergic neurons within the substantia nigra (SN). While the precise etiology of dopaminergic neuronal demise is elusive, multiple lines of evidence indicate that neuroinflammation is involved in the pathogenesis of PD. We have previously demonstrated that subcutaneous administration of bee venom (BV) phospholipase A 2 (bvPLA 2 ) suppresses dopaminergic neuronal cell death in a PD mouse model. In the present study, we established standardized methods for producing bvPLA 2 agent isolated from crude BV at good manufacturing practice (GMP) facility. The therapeutic efficacy of purified bvPLA2 agent was examined in MPTP-induced PD mice. Importantly, administration of purified bvPLA 2 in a dose-dependent manner reversed motor deficits in PD mice as well as inhibited loss of dopaminergic neurons within the SN of PD mice. The concentration-dependent action of standardized bvPLA 2 appeared to be related to the induction of CD4 + CD25 + Foxp3 + regulatory T cells (Tregs), which, in part, inhibits T helper 1 (Th1) and Th17 polarization and suppresses microglial activation in PD mice. Taken together, these results suggest that standardized bvPLA 2 purified from BV shows a neuroprotective effect against PD and thus has a potential target for treatment of PD.

Background: Open distal graft to proximal aortic arch anastomosis is central to a hemiarch replacement. Even if the ischemic tolerance time of several organs during circulatory arrest (CA) at normothermia is much longer than that of the brain, very little is known about the safety and clinical efficacy of moderate hypothermia for organ protection during the average duration of CA needed for aortic arch replacement. Hemiarch replacement using the standard techniques of deep hypothermia and antegrade perfusion has often ignored the effects of prolonged distal body CA. Maintenance of distal organ, especially the liver and kidney, perfusion reduces the risk of postoperative renal dysfunction and coagulopathy., Case Presentation: A 72-year-old female patient was referred to our institute due to chest discomfort. Radiologic investigations revealed a giant aneurysm of the ascending aorta extending but confined to the proximal aortic arch. We performed an alternative technique for hemiarch replacement using a dual inflow source., Conclusions: Although this technique cannot apply to all aneurysmal aortic diseases, our basic technique involving the use of dual inflow may be well suited for standard hemiarch replacement that is confined to the proximal aortic arch, given the shortening of the bypass and ischemic times.

Background: An aberrant right subclavian artery (ARSA) is a relatively prevalent vascular anomaly. What is the most appropriate treatment for thoracic aortic aneurysm combined a non-aneurysmal change ARSA?, Case Presentation: A 52-year-old man was admitted to our institute due to a history of chronic cough, dysphagia and an abnormal chest radiographic finding. Because of his progressive symptoms and large fusiform thoracic aneurysm, we performed the hybrid repair for simultaneous relief of an ARSA causing dysphagia and thoracic aneurysm., Conclusion: In case without aneurysm of ARSA, especially in conjunction with approximate thoracic aneurysm, our approach is suitable because the revascularization using the right carotid to subclavian artery re-routing prior to endograft deployment is justified in order to preserve circulation of posterior brain, spinal cord, internal mammary artery and upper limb and to prevent large retrograde type II endoleaks, as well as simplicity and durability.

Idiopathic pulmonary fibrosis (IPF) is a devastating and common chronic lung disease pathologically characterized by loss of epithelial cells and activation of fibroblasts and myofibroblasts. The etiology of IPF remains unclear and the disease pathology is poorly understood with no known efficacious therapy. PM014 is an herbal extract that has been shown to have beneficial effects in pulmonary diseases, which are likely to exert anti-inflammatory bioactions. In the present study, we observed that bleomycin (BLM) caused increased inflammatory infiltration as well as collagen deposition in lungs of mice on day 14 after treatment. Administration of PM014 suppressed BLM-induced inflammatory responses and fibrotic changes in dose-dependent manner in mice. Additionally, we provided in vitro evidence suggesting that PM014 inhibited TGF-β1-induced epithelial-mesenchymal transition (EMT) and fibroblast activation in alveolar epithelial cells and human lung fibroblasts from healthy donor and IPF patients. PM014 appeared to target TGF-β1 signaling via Smad-dependent pathways and p38 mitogen-activated protein kinases (MAPKs) pathways. Taken together, our data suggest that PM014 administration exerts a protective effect against lung fibrosis and highlight PM014 as a viable treatment option that may bring benefits to patient with IPF.

Cellular homeostasis is coordinated through communication between mitochondria and the nucleus, organelles that each possess their own genomes. Whereas the mitochondrial genome is regulated by factors encoded in the nucleus, the nuclear genome is currently not known to be actively controlled by factors encoded in the mitochondrial DNA. Here, we show that MOTS-c, a peptide encoded in the mitochondrial genome, translocates to the nucleus and regulates nuclear gene expression following metabolic stress in a 5'-adenosine monophosphate-activated protein kinase (AMPK)-dependent manner. In the nucleus, MOTS-c regulated a broad range of genes in response to glucose restriction, including those with antioxidant response elements (ARE), and interacted with ARE-regulating stress-responsive transcription factors, such as nuclear factor erythroid 2-related factor 2 (NFE2L2/NRF2). Our findings indicate that the mitochondrial and nuclear genomes co-evolved to independently encode for factors to cross-regulate each other, suggesting that mitonuclear communication is genetically integrated., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Despite recent advances in surgical techniques and perioperative management, the surgical mortality of acute type A aortic dissection remains high., Objective: In a hospital with few acute type A aortic dissection operations, we retrospectively investigated whether simplified surgical approach could obtain proper surgical outcomes., Methods: Between October 2007 and December 2016, we performed emergency surgery in 99 patients who had acute type A aortic dissection, including replacement of the hemi-arch in 62 patients (63%) and total arch in 32 patients (32%) and aortic root surgery in 7 patients (7%). Surgical strategy has been simplified over time., Results: We performed right axillary artery perfusion in 61 patients (62%) and antegrade cerebral perfusion in 78 patients (79%). During the last 3 years, in-hospital mortality was decreased to 4% (2/47). Preoperative unresolved shock was an independent predictor of hospital death. Although the patients with total arch replacement or aortic root surgery had a mean significantly long cardiopulmonary bypass or circulatory arrest time, in-hospital mortality or neurologic complications was not increased., Conclusions: Simplified surgical approach could provide a reasonable surgical outcome in acute type A aortic dissection surgery in a center with a small volume of acute aortic dissection surgery.

Mitochondria are ancient organelles that are thought to have emerged from once free-living α-proto-bacteria. As such, they still possess several bacterial-like qualities, including a semi-autonomous genetic system, complete with an independent genome and a unique genetic code. The bacterial-like circular mitochondrial DNA (mtDNA) has been described to encode 37 genes, including 22 tRNAs, 2 rRNAs, and 13 mRNAs. Two additional peptides reported to originate from the mtDNA, namely humanin (Hashimoto et al., 2001; Ikone et al., 2003; Guo et al., 2003) [1-3] and MOTS-c (mitochondrial ORF of the twelve S c) (Lee et al., 2015) [4], indicate a larger mitochondrial genetic repertoire (Shokolenko and Alexeyev, 2015) [5]. These mitochondrial-derived peptides (MDPs) have profound and distinct biological activities and provide a paradigm-shifting concept of active mitochondrial-encoded signals that act at the cellular and organismal level (i.e. mitochondrial hormone) (da Cunha et al., 2015; Quiros et al., 2016) [6,7]. Considering that mitochondria are the single most important metabolic organelle, it is not surprising that these MDPs have metabolic actions. MOTS-c has been shown to target the skeletal muscle and enhance glucose metabolism. As such, MOTS-c has implications in the regulation of obesity, diabetes, exercise, and longevity, representing an entirely novel mitochondrial signaling mechanism to regulate metabolism within and between cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

After tricuspid valve surgery for long-standing tricuspid regurgitation associated with right ventricular failure, reverse remodelling of the enlarged right ventricle, including recovery of right ventricular systolic function, is unpredictable. We present the case of a 31-year old man with early reduction of dilated right ventricular dimensions and delayed recovery of impaired right ventricular systolic function after valve repair for traumatic tricuspid regurgitation lasting 16 years., (© The Author 2015. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Valve replacement is typically the most appropriate option for treating aortic valve stenotic insufficiency. However, neither mechanical nor bioprosthetic replacement components preserve the circumferential expansion and contraction of a native aortic annulus during the cardiac cycle, because the prosthetic ring is affixed to the annulus. A 64-year-old man presented with a bicuspid and stenotic aortic valve, and the native annulus was too small to accommodate a porcine replacement valve. We fashioned new aortic leaflets from bovine pericardium with use of a template, and we affixed the sinotubular junction with use of inner and outer stabilization rings. Postoperative echocardiograms revealed coaptation of the 3 new leaflets with no regurgitation. At the patient's 5.5-year follow-up examination, echocardiograms showed flexible leaflet movement with a coaptation height of 7 mm, and expansion and contraction of the aortic annulus similar to that of a normal native annulus. The transvalvular pressure gradient was insignificant. If long-term durability of the new leaflets is confirmed, this method of leaflet replacement and fixation of the sinotubular junction might serve as an acceptable alternative to valve replacement in the treatment of aortic valve stenosis. We describe the patient's case and present our methods and observations.

Objective: DNA methylation, a regulator of gene expression, plays an important role in diverse biological processes including developmental process, carcinogenesis and aging. In particular, aberrant DNA methylation has been largely observed in several types of cancers. Currently, it is important to extract disease-specific gene sets associated with the regulation of DNA methylation., Materials and Methods: Here we propose a novel approach to find the minimum regulatory units of genes, co-methylated and co-expressed gene pairs (MEGP) that are highly correlated gene pairs between DNA methylation and gene expression showing the co-regulatory relationship. To evaluate whether our method is applicable to extract disease-associated genes, we applied our method to a large-scale dataset from the Cancer Genome Atlas extracting significantly associated MEGP and analyzed their functional correlation., Results: We observed that many MEGP physically interacted with each other and showed high semantic similarity with gene ontology terms. Furthermore, we performed gene set enrichment tests to identify how they are correlated in a complex biological process. Our MEGP were highly enriched in the biological pathway associated with ovarian cancers., Conclusions: Our approach is useful for discovering coordinated epigenetic markers associated with specific diseases.

To explore the physiological significance of N-glycan maturation in the plant Golgi apparatus, gnt1, a mutant with loss of N-acetylglucosaminyltransferase I (GnTI) function, was isolated in Oryza sativa. gnt1 exhibited complete inhibition of N-glycan maturation and accumulated high-mannose N-glycans. Phenotypic analyses revealed that gnt1 shows defective post-seedling development and incomplete cell wall biosynthesis, leading to symptoms such as failure in tiller formation, brittle leaves, reduced cell wall thickness, and decreased cellulose content. The developmental defects of gnt1 ultimately resulted in early lethality without transition to the reproductive stage. However, callus induced from gnt1 seeds could be maintained for periods, although it exhibited a low proliferation rate, small size, and hypersensitivity to salt stress. Shoot regeneration and dark-induced leaf senescence assays indicated that the loss of GnTI function results in reduced sensitivity to cytokinin in rice. Reduced expression of A-type O. sativa response regulators that are rapidly induced by cytokinins in gnt1 confirmed that cytokinin signaling is impaired in the mutant. These results strongly support the proposed involvement of N-glycan maturation in transport as well as in the function of membrane proteins that are synthesized via the endomembrane system., (© 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.)

Endocarditis after mitral valve (MV) annuloplasty is uncommon. The ring used in MV annuloplasty is often inadequate because it opposes the growth of the MV leaflets and annulus. We report a 15-year-old male that required redo surgery for prosthetic ring endocarditis 40 months after a previous MV annuloplasty. After the previous ring was removed, the undergrown posterior leaflet was repaired with pericardial augmentation and the posterior annulus was stabilized with a Mitra-Lift® supra-annular strip to preserve a flexible valve orifice and allow the anterior MV annulus and the commissures to grow in relation to body size., (© 2012 Wiley Periodicals, Inc.)

We describe a technique for treating severe functional tricuspid regurgitation (TR) when residual regurgitation cannot be eliminated with ring annuloplasty alone. The anterior leaflet and the anterior half of the posterior leaflet are augmented with an elliptic pericardial patch before implantation of a rigid annuloplasty ring. We successfully performed this procedure in 9 patients with severe TR due to severe leaflet tethering or short coaptation length and achieved complete elimination of TR with sufficient coaptation length in tricuspid valve leaflets for all patients., (Copyright © 2011 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Objective: The purpose of this study was to examine the effect of neuropathic pain produced by peripheral nerve damage on mass, myofibrillar protein content, and cross-sectional areas of Type I and II fibers of rat hindlimb muscles., Method: Adult male Sprague-Dawley rats were assigned to one of three groups: a pain group (n = 10) that underwent ligation and cut of the left L5 spinal nerve, a sham group (n = 10) that underwent a sham cut procedure, or a control group (n = 10) that underwent no procedures. The withdrawal threshold test was done to assess pain threshold on each of Days 1-7 and 14. Activity, body weight, and food intake were measured daily for 2 weeks. At 15 days, rats were anesthetized and the bilateral soleus, plantaris, and gastrocnemius muscles dissected., Results: At 15 days post-ligation, the pain group had significant decreases in total dietary intake, body weight, activity, and muscle weight as compared to sham and control animals. Muscle weight and cross-sectional area of Type II fiber of the ipsilateral soleus, plantaris, and gastrocnemius muscles decreased as did myofibrillar protein content of the ipsilateral plantaris and gastrocnemius muscles. Muscle weight of the contralateral gastrocnemius muscle decreased, as did myofibrillar protein content and cross-sectional area of Type II fiber of the contralateral plantaris muscle., Conclusion: Hindlimb muscle atrophy occurs in both ipsilateral and contralateral sides following induction of neuropathic pain by unilateral peripheral nerve damage. Muscle changes of the ipsilateral side are more pronounced than those of the contralateral side.

Most medical literature regarding the anomalous origin of a coronary artery from the opposite sinus of Valsalva pertains to sudden death in the young. The surgical treatment of anomalous origin of the left main coronary artery from the right sinus of Valsalva is not particularly well codified, and when an anomalous left main coronary artery is associated with an extramural stenosis, treatment becomes more technically challenging. Herein, we describe a unique approach toward treating this combination of conditions. A 15-year-old adolescent boy was diagnosed with anomalous origin of the left main coronary artery from the right sinus of Valsalva. The condition was accompanied by a slit ostium and an extramural stenosis of the left main coronary artery. Surgery was offered to the patient in view of his young age and the uncertain prospective course of the disease. Coronary unroofing was not indicated, due to the extramural location of the stenosis. With the patient under cardiopulmonary bypass, the stenotic segment of the left main coronary artery was transected outside the aorta, repaired by vein patch augmentation, and reimplanted directly into the left coronary sinus. The patient recovered uneventfully. A postoperative computed tomographic angiogram showed good patency of the reconstructed artery. We expect excellent longevity of the directly reimplanted coronary artery.

This study was performed to determine which of uninjured lumbar 4 (L4) C-afferents and injured L5 afferents was important for the generation of mechanical hypersensitivity following L5 spinal nerve ligation-and-cut (SNLC, modified spinal nerve ligation) in the rat. The mechanical hypersensitivity established following L5 SNLC was completely abolished 6 weeks after local capsaicin treatment of the sciatic nerve or L4 spinal nerve. At this stage, a substantial number of capsaicin-sensitive C-afferents were eliminated without any loss of A-afferents in the L4 spinal segment, suggesting that the capsaicin-sensitive L4 C-afferents are a major contributor to L5 SNLC-produced mechanical hypersensitivity. The peripheral terminals of L4 C-afferents are active in maintaining mechanical hypersensitivity, even long after L5 SNLC. When capsaicin-sensitive L4 C-afferents were previously eliminated, the induction of L5 SNLC-produced hypersensitivity was partly prevented. Thus, capsaicin-sensitive L4 C-afferents are crucial for the induction and maintenance of mechanical hypersensitivity in the L5 SNLC model. Also, when capsaicin-sensitive L4 C-afferents were previously eliminated, L5 SNLC still produced a partial mechanical hypersensitivity for a 1- to 2-week maintenance period with a several-day delay. This mild hypersensitivity was prevented by the previous L5 dorsal rhizotomy, implying an involvement of inputs from injured L5 afferents in the maintenance of hypersensitivity at the earlier stage. The results suggest that uninjured C-afferents, most likely C-polymodal nociceptors, are necessary for the induction and maintenance of neuropathic pain, and that afferent inputs, presumably from injured Abeta-fibers, also contribute to the maintenance at an earlier stage.

An electrospinning method was used to fabricate chitin nanofibrous matrices for biodegradability and cell behavior tests. The morphology of as-spun chitin nanofibers (Chi-N) and commercial chitin microfibers (Beschitin W; Chi-M) was investigated by scanning electron microscopy. From the image analysis, the average diameters of Chi-N and Chi-M were 163 nm and 8.77 microm, respectively. During in vitro degradation for 15 days, the degradation rate of Chi-N was faster than that of Chi-M, likely due to higher surface area of Chi-N. Chi-N that was grafted into rat subcutaneous tissue had almost degraded within 28 days, and no inflammation could be seen on the nanofiber surfaces or in the surrounding tissues (except in the early stage wound). To assay and compare the cytocompatibility and cell behavior with Chi-N and Chi-M, cell attachment and spreading of normal human keratinocytes and fibroblasts seeded on chitin matrices and the interaction between cells and chitin fibers were studied. Relatively high cell attachment and spreading of all the cells tested were observed on Chi-N in comparison to Chi-M, and Chi-N treated with type I collagen significantly promoted the cellular response. Our results indicate that the Chi-N, alone or with extracellular matrix proteins (particularly type I collagen), could be potential candidates for the cell attachment and spreading of normal human keratinocytes and fibroblasts. This property of Chi-N might be particularly useful for wound healing and regeneration of oral mucosa and skin.

BMP-2 (bone morphogenetic protein-2) has been used in promoting bone formation and, in combination with various delivery carriers, in bone regeneration or bone tissue-engineering practice. In the present study, BMP-2 was immobilized directly on a GBR (guided bone-regenerative) membrane surface made of chitosan nanofibres, providing a bioactive surface that can enhance bone-regeneration capacity. The BMP-2-conjugated membrane surface retained bioactivity for up to 4 weeks of incubation, as well as holding over 50% of the initial BMP-2 attached. The BMP-2-conjugated surface increased osteoblastic cell attachment in accordance with the amount of BMP-2 conjugated. In addition, the BMP-2-conjugated chitosan membrane significantly promoted cell proliferation, alkaline phosphatase activity, as well as calcium deposition when compared with BMP-2-adsorbed membrane, indicating a stable localization of BMP-2 at the surface of the chitosan membrane. Taking these findings together, the conjugation of BMP-2 on a chitosan nanofibrous membrane would seen to be applicable for inducing significant and localized bone formation in future GBR procedures.

Injectable polysaccharide microcapsules holding minocycline were fabricated from alginate and chitosan for the treatment of periodontitis. The microcapsules were examined for the release and degradation of minocycline, as well as antimicrobial activity. The microcapsules were biodegradable and released minocycline between 10 and 1000 microg ml(-1), which was higher than the usual therapeutic concentration (1-5 microg ml(-1)), for up to 7 days. These microcapsules showed a statistically significant suppression of pathogenic bacteria, such as Prevotella intermedia causing periodontitis. The microcapsules are thus potentially useful for drug delivery for the treatment of periodontitis.

Purpose: A specially designed self-expandable covered metallic stent incorporated with beta-emitting radioisotope, Holmium-166 (Ho-166), was developed for delivering intraluminal brachytherapy as well as for internal bile drainage in malignant biliary stricture. The purpose of the study was to demonstrate the safety and tissue response of the radioactive metallic stent on the normal canine common bile duct (CBD) prior to the clinical application., Materials and Methods: Nitinol self-expandable stents (diameter; 4 mm, length; 20 mm) were covered with polyurethane membrane (50 microm thick) containing 21 to 135 muCi of Ho-166 (mean, 77.9 microCi). To prevent migration of stent, the membrane covered only the middle 1 cm of the stent and the ends of the stent were left uncovered. The stents were placed in the CBD of 20 healthy beagle dogs. For control, non-radioactive covered stents were placed in another three dogs. The dogs were killed 3 to 6 months after stent insertion and histopathologic examination of CBD was performed., Results: There was no stent migration in all cases. Varying degrees of papillary mucosal hyperplasia leading to significant narrowing of the lumen was observed within the lumen of the bare portion and under the mucosal surface of the non-radioactive covered stents. However, fibrosis was noted in Ho-166 coated area, instead of mucosal hyperplasia. Severity of fibrosis was correlated with estimated radiation dose. Despite high dose, there was no perforation of CBD wall. The membrane of Ho-166 coat was disrupted in some cases of 6 months follow-up., Conclusions: Holmium-166 incorporated covered stents demonstrated fibrosis of CBD wall and inhibition of ingrowth of mucosal hyperplasia without serious complication such as perforation, while control group showed severe mucosal hyperplasia.

Arylsulfatase cloned from a marine aerobic Gram-negative bacterium, Pseudoalteromonas carrageenovora, was overexpressed in Escherichia coli with 10 microM IPTG induction. The expressed recombinant arylsulfatase was purified to homogeneity from the harvested cells through osmotic disruption and column chromatography methods, such as DEAE-cellulose anion exchange chromatography and Heparin-Sepharose affinity chromatography. The purified arylsulfatase was kinetically characterized using the synthetic substrate of phenolic ester, p-nitrophenyl sulfate (pNPS). One unit of arylsulfatase catalyzes the liberation of 1.0 micromol p-nitrophenol from pNPS per minute. The purified enzyme has a specific activity of 468 U/mg with a purification yield of 27% from the cell lysate, and exhibited an estimated molecular mass of 33 kDa in SDS-PAGE analysis. The precursor polypeptide of 36 kDa was processed by releasing a putative signal peptide, and the mature arylsulfatase of 33.1 kDa with a N-terminal sequence of S-E-T-K-N was trafficked to periplasmic space. The enzyme had optimum reaction conditions for activity at pH 7.0 and at a temperature range of 40-45 degrees C. The apparent K(M) and k(cat) of the enzyme for hydrolysis of pNPS at pH 7.0 and at 45 degrees C were determined to be 1.15 mM and 1000 s-1, respectively. Based on inhibitor studies along with optimal pH values and preferential periplasmic location of the enzyme, we suggest that the recombinant arylsulfatase from P. carrageenovora is probably similar to the Klebsiella sulfatase with serine residue in the active site.

A simple procedure was developed for coating the surface of a conventional percutaneous transluminal coronary angioplasty balloon angioplasty catheter with 166Ho. The absorbed dose delivered by the surface-coated catheter is twice that of a similar catheter filled with a solution containing the same activity of 166Ho. Leakage tests show that <0.6% of the coated activity is removable from the surface of the catheter. The coated catheter removes the risk of release of the 166Ho into the body as a result of rupture of the balloon, and also reduces the radiation exposure to staff.

SEDL is an evolutionarily highly conserved protein in eukaryotic organisms. Deletions or point mutations in the SEDL gene are responsible for the genetic disease spondyloepiphyseal dysplasia tarda (SEDT), an X-linked skeletal disorder. SEDL has been identified as a component of the transport protein particle (TRAPP), critically involved in endoplasmic reticulum-to-Golgi vesicle transport. Herein, we report the 2.4 A resolution structure of SEDL, which reveals an unexpected similarity to the structures of the N-terminal regulatory domain of two SNAREs, Ykt6p and Sec22b, despite no sequence homology to these proteins. The similarity and the presence of unusually many solvent-exposed apolar residues of SEDL suggest that it serves regulatory and/or adaptor functions through multiple protein-protein interactions. Of the four known missense mutations responsible for SEDT, three mutations (S73L, F83S, V130D) map to the protein interior, where the mutations would disrupt the structure, and the fourth (D47Y) on a surface at which the mutation may abrogate functional interactions with a partner protein.

Intratumoural injection of an unsealed beta-emitting radionuclide is a new technique for the local control of tumours that has the advantage of delivering a higher radiation dose to tumour while minimising radiation hazard to the surrounding normal tissues. In this study, therapeutic effect, morphological alterations and biological responses to the high-dose continuous irradiation delivered using this new technique were evaluated in an animal model with B16 melanoma. For evaluation of the therapeutic effect, 92 C57BL/6 mice with B16 melanoma were divided into four groups. In each group, intratumoural injections were performed when the tumour measured approximately 1 cm along its long axis. Group 1 (n=25) received 0.3 ml of normal saline, group 2 (n=15) 37 MBq of carrier-free holmium-166 in 0.3 ml saline, group 3 (n=27) 185 MBq of 166Ho in 0.3 ml saline and group 4 (n=25) 185 MBq of 166Ho in 0.5 ml saline. In addition, another 30 mice were used for morphological and biological analysis of the radiation effect. These 30 mice were injected with 185 MBq of 166Ho in 0.3 ml saline, and five were sacrificed at each of the following six time points: before injection and 1, 2, 3, 6 and 14 days post injection. Haematoxylin-eosin (H&E) staining, immunohistochemical analysis for p53, p21, PCNA and cyclin D1, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) staining, reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry were performed. For visual side-by-side comparison, melanoma cells were inoculated bilaterally into the back of ten additional mice, and 185 MBq of 166Ho in 0.3 ml of saline or an equal volume of normal saline was injected separately into the bilateral tumours. Nine days after inoculation of melanoma cells, mean tumour volume reached 492.5-631.9 mm3. Tumours of the control group (group 1) showed rapid growth, and the mean tumour volume reached approximately 30 times the original volume. None of the control group lived for more than 16 days following the injection of normal saline. On the other hand, mean tumour volume of the treated groups showed a gradual decrease, and 67%-74% of the treated animals were alive when all the control animals had died. The median survival of the control group was 9 days following injection, whereas it was 29 days in group 2, 33 days in group 3 and 33 days in group 4. The survival rate of group 3 was higher than that of groups 2 and 4, but statistical significance was not observed. H&E stain of the tumours demonstrated central necrosis and peripheral residual cells with progressive cytoplasmic and nuclear swelling without apoptotic features. Expression of proteins and mRNAs of p53 and bax increased until 3 days, as compared with 48 h for p21; thereafter, the expression gradually decreased. TUNEL-positive nuclei could be seen from 2 days until 2 weeks after treatment. Flow cytometry did not demonstrate an increase in apoptotic features as compared with the control animals. In conclusion, intratumoural injection of the unsealed beta-emitting radionuclide 166Ho appears to be a promising alternative radiotherapeutic modality for the local control of malignant melanoma. The main cell death mechanisms with this technique seem to be radiation-induced central necrosis and peripheral growth arrest or secondary necrosis of tumour cells, rather than apoptosis.