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Fetal cardiomyocytes actively proliferate to form the primitive heart in utero in mammals, but they stop dividing shortly after birth. The identification of essential molecules maintaining this active cardiomyocyte proliferation is indispensable for potential adult heart regeneration. A recent study has shown that this proliferation depends on a low fetal oxygen condition before the onset of breathing at birth. We have established an isolation protocol for mouse fetal cardiomyocytes, performed under strict low oxygen conditions to mimic the intrauterine environment, that gives the highest proliferative activities thus far reported. Oxygen exposure during isolation/culture markedly inhibited cell division and repressed cell cycle-promoting genes, and subsequent genome-wide analysis identified Fam64a as a novel regulatory molecule. Fam64a was abundantly expressed in hypoxic fetal cardiomyocyte nuclei, but this expression was drastically repressed by oxygen exposure, and in postnatal cardiomyocytes following the onset of breathing and the resulting elevation of oxygen tension. Fam64a knockdown inhibited and its overexpression enhanced cardiomyocyte proliferation. Expression of a non-degradable Fam64a mutant suggested that optimum Fam64a expression and subsequent degradation by anaphase-promoting complex/cyclosome (APC/C) during the metaphase-to-anaphase transition are required for fetal cardiomyocyte division. We propose that Fam64a is a novel cell cycle promoter of hypoxic fetal cardiomyocytes in mice.

The use of viral vectors for inner ear gene therapy is receiving increased attention for treatment of genetic hearing disorders. Most animal studies to date have injected viral suspensions into neonatal ears, via the round window membrane. Achieving transduction of hair cells, or sensory neurons, throughout the cochlea has proven difficult, and no studies have been able to efficiently transduce sensory cells in adult ears while maintaining normal cochlear function. Here, we show, for the first time, successful transduction of all inner hair cells and the majority of outer hair cells in an adult cochlea via virus injection into the posterior semicircular canal. We used a “designer” AAV, AAV2/Anc80L65, in which the main capsid proteins approximate the ancestral sequence state of AAV1, 2, 8, and 9. Our injections also transduced ~10% of spiral ganglion cells and a much larger fraction of their satellite cells. In the vestibular sensory epithelia, the virus transduced large numbers of hair cells and virtually all the supporting cells, along with close to half of the vestibular ganglion cells. We conclude that this viral vector and this delivery route hold great promise for gene therapy applications in both cochlear and vestibular sense organs.

The use of viral vectors for inner ear gene therapy is receiving increased attention for treatment of genetic hearing disorders. Most animal studies to date have injected viral suspensions into neonatal ears, via the round window membrane. Achieving transduction of hair cells, or sensory neurons, throughout the cochlea has proven difficult, and no studies have been able to efficiently transduce sensory cells in adult ears while maintaining normal cochlear function. Here, we show, for the first time, successful transduction of all inner hair cells and the majority of outer hair cells in an adult cochlea via virus injection into the posterior semicircular canal. We used a “designer” AAV, AAV2/Anc80L65, in which the main capsid proteins approximate the ancestral sequence state of AAV1, 2, 8, and 9. Our injections also transduced ~10% of spiral ganglion cells and a much larger fraction of their satellite cells. In the vestibular sensory epithelia, the virus transduced large numbers of hair cells and virtually all the supporting cells, along with close to half of the vestibular ganglion cells. We conclude that this viral vector and this delivery route hold great promise for gene therapy applications in both cochlear and vestibular sense organs.

Recent studies have indicated a high prevalence of persistent postoperative pain (PPP) in patients who undergo various surgical procedures. Patient suffering from PPP and the cost to manage this pain are substantial socioeconomic burdens. Although recent studies have revealed various underlying mechanisms of PPP, prevention and management remains unsatisfactory. In this review, we briefly summarize the current understanding of etiology and pathophysiology, which include neuropathic, nociceptive, inflammatory, and myofascial pains as well as psychological or psychiatric factors, and advances in the prevention and management of PPP over the last decade. In addition to the literature review, we present findings from our observation studies of patients with post-thoracotomy pain syndrome, which indicate myofascial pain as a major pathophysiological cause of PPP. Based on the literature review and our recent clinical studies, we propose a comprehensive practical strategy for the prevention and management of PPP.

We describe a case of urinary retention caused by viral sacral myeloradiculitis (Elsberg syndrome) that occurred one week after spinal anesthesia. The differential diagnosis of urinary retention after spinal anesthesia is discussed.A 76-yr-old male patient presented for operative removal of a right testicular hydrocele under spinal anesthesia. Anesthesia and surgery were uneventful, and he was discharged on the fifth postoperative day. Two days after discharge, he developed intermittent anal pain and voiding difficulty and was readmitted to hospital on the tenth postoperative day. He subsequently developed urinary retention, incontinence of feces, and difficulty in defecation. Magnetic resonance imaging showed no epidural hematoma, abscess, or other lesions in the spinal column, cauda equina, or spinal cord. Neurological examination showed dysesthesia in the perineal region and loss of the anal reflex and bulbocavernosus response, which indicated sacral (S4-5) radiculopathy or a lesion of the conus of the spinal cord. A cerebrospinal analysis showed slight elevation of protein without pleocytosis. After neurologic consultation, herpetic sacral myeloradiculitis was suspected and intravenous acyclovir was administered along with large doses of methylprednisolone and immunoglobulin. The symptoms gradually resolved, and the difficulty in voiding resolved 19 days after initiation of the treatment. The patient was discharged 23 days after the start of the treatment without any other complications.This case suggests that Elsberg syndrome is important in the differential diagnosis of urinary retention after spinal anesthesia and should be discriminated from other anesthesia-related complications.

Objective: Clear cell carcinoma (CCC) is a rare, low-grade malignant neoplasm of minor salivary glands. Clear cell carcinoma is composed of monomorphic clear cells that contain glycogen within a hyalinized stroma. The microscopic diagnosis can be challenging, and the treatment strategy has not been established. Methods: We report a case of a 56-year-old woman with CCC in the base of the tongue. A literature review, including 22 published cases and our case, was performed to clarify the clinical characteristics of CCC in the base of the tongue. Results: The exophytic tumor showed no infiltration to the neighboring tissues. She underwent a local excision en bloc transorally and is free of the disease 15 months after surgery. In our review of 23 cases of CCC in the base of the tongue, 21 cases received surgical excision for the purpose of the primary treatment modality. The fact that the distinctive features of restricted site-related symptoms and the rare presentation of pain emerged was similar to previously reported cases of CCC. A relatively good prognosis was still indicated. Conclusion: We reveal the features of the unusual occurrence of CCC in the base of the tongue and consider the appropriate surgical approach for primary treatment.

In recent years, the authors have been conducting research on the establishment of technical prevention measures for development and design utilizing highly-accurate CAE analysis. This research investigates the mechanisms that cause nut loosening, which is a matter of concern for automobile manufacturers around the world. Specifically, actual machine tests were conducted using three types of bolt and nut fasteners with

Royal jelly (RJ), the exclusive food for queen bees, is taken as a dietary supplement because it is highly rich in nutrients. However, RJ is known to induce an anaphylactic response in some individuals. We evaluated in the present study the hypoallergenicity of alkaline protease-treated RJ in vitro and in vivo. We first confirmed that this treated RJ contained the same levels of vitamins, minerals and specific fatty acid as in untreated RJ. We then showed that the IgE-binding capacity of the treated RJ was very significantly reduced by conducting in vitro assays of the blood from RJ-sensitive patients. An in vivo skin-prick test on the RJ-sensitive patients also showed that, in the majority of the patients (3 out of 4 tested), the treated RJ did not evoke any allergenic response. It is thus advantageous to prepare hypoallergenic RJ by a protease enzyme treatment for its safe consumption.

Study design We measured the response of the behavior and spinal glial activation to anti-nerve growth factor receptor (p75 neurotrophin receptor [p75NTR]) antibodies in the rat brachial plexus avulsion (BPA) model. Objective The aim of this study was to investigate the effect of anti-p75NTR antibodies on nociceptive behavior and activation of spinal microglia in the rat BPA model. Summary of background data Tanezumab (anti-nerve growth factor antibody) treatment is associated with pain reduction and improvement in function, but with several complications. Methods Thirty male Wistar rats were used. In the BPA group, the C8-T1 roots were avulsed from the spinal cord with forceps at the lower trunk level and 10 μL of saline was applied locally (n = 10). In the anti-p75NTR group, the C8-T1 roots were avulsed and 10 μL of anti-p75NTR antibody was applied locally (n = 10). In a sham-operated group, the lower trunk was simply exposed (n = 10). Mechanical hyperalgesia and pain-induced walking patterns were measured using von Frey filaments (Stoelting, Wood Dale, IL) and the CatWalk gait analysis (Noldus Information Technology, the Netherlands) system every third day for 3 weeks. Activation of astrocytes and microglia was immunohistochemically examined in the spinal cord using anti-glial fibrillary acidic protein (GFAP) and anti-Iba1 antibodies both 7 and 21 days after surgery. Results Animals in the BPA group displayed significant mechanical hyperalgesia that continued through day 21 compared with animals in the sham-operated group, and mechanical hyperalgesia in the anti-p75NTR group was significantly improved 6 days after the operation. Regarding pain-induced gait analysis via CatWalk, animals in the BPA group displayed a significantly greater pain-like gait pattern than the p75 group for up to 3 weeks. Levels of GFAP-immunoreactive astrocytes and Iba1-immunoreactive microglia in the anti-p75NTR group were significantly reduced compared with the BPA group. Conclusion Our results suggest that p75NTR contributes to neuropathic pain associated with BPA, and that inhibition of p75NTR reduces neuropathic pain. Level of evidence N/A.

The effective uptake of antigens (Ags) by specialized M cells of gut‐associated lymphoid tissues is an important step in inducing an efficient intestinal mucosal immune response. In this study, royal jelly (RJ) was found to stimulate the differentiation of M‐like cells from human Caco‐2 cells in an in vitro M cell model. Furthermore, RJ and protease‐treated royal jelly (pRJ) efficiently enhanced transcytosis of FluoSpheres® carboxylate‐modified microspheres from the apical side to the basolateral side in the model. Therefore, we evaluated the ability of pRJ to induce efficient mucosal immune responses in an in vivo nonhuman primate. Continuous oral administration of commercially available pRJ resulted in a significant enhacement of the antigen‐specific IgA response in stool sample. Interestingly, Caco‐2 monolayer assay demonstrated that ether extracts from pRJ efficiently increased the expression level of a universal M cell marker, glycoprotein 2 (gp2). These findings suggest that pRJ exhibits mucosal immunomodulatory properties via stimulation of effective uptake of Ags through M cells.

In Guatemala, the Ministry of Health (MoH) began a vector control project with Japanese cooperation in 2000 to reduce the risk of Chagas disease infection. Rhodnius prolixus is one of the principal vectors and is targeted for elimination. The control method consisted of extensive residual insecticide spraying campaigns, followed by community-based surveillance with selective respraying. Interventions in nine endemic departments identified 317 villages with R. prolixus of 4,417 villages surveyed. Two cycles of residual insecticide spraying covered over 98% of the houses in the identified villages. Fourteen villages reinfestated were all resprayed. Between 2000-2003 and 2008, the number of infested villages decreased from 317 to two and the house infestation rate reduced from 0.86% to 0.0036%. Seroprevalence rates in 2004-2005, when compared with an earlier study in 1998, showed a significant decline from 5.3% to 1.3% among schoolchildren in endemic areas. The total operational cost was US$ 921,815, where the cost ratio between preparatory, attack and surveillance phases was approximately 2:12:1. In 2008, Guatemala was certified for interruption of Chagas disease transmission by R. prolixus. What facilitated the process was existing knowledge in vector control and notable commitment by the MoH, as well as political, managerial and technical support by external stakeholders.

Influenza is one of the important respiratory tract infections that require special attention for maintaining health and hygiene. The removal of influenza virus (IFV) by secretory IgA produced by the respiratory epithelium has been reported to be a critical host defense mechanism. Therefore, we isolated Lactobacillus kunkeei YB38 (YB38), the promoter of the salivary IgA secretion in humans, from honeybee pollen and studied the effect of heat-killed YB38 treatment for preventing IFV infection in a mouse model. Female BALB/c mice received YB38 orally for 21 consecutive days and were then inoculated nasally with IFV. The YB38-treated group with a daily dose of 100 mg/kg showed an increased survival rate after IFV infection relative to the control. IgA secretion in the respiratory epithelium in the YB38-treated group (100 mg/kg) was significantly increased after 6 days of infection, while IL-6 production in the same respiratory site and the number of cells infiltrating into alveoli were significantly decreased. Moreover, lung tissue damage that appeared after IFV infection was reduced. These results suggested that the YB38 dose induced early and local IgA secretion at the infection site, inhibited persistent IFV infection, and prevented the infiltration of inflammatory immune cells or production of excessive IL-6, resulting in less damage to lung tissues.

Tyrosinase is the key enzyme involved in melanogenesis. The aim of this study was to investigate the in vitro inhibitory effects of gnetin C, a resveratrol dimer isolated from melinjo (Gnetum gnemon) seeds, on tyrosinase activity and melanin biosynthesis in murine B16 cells. The inhibitory activities of gnetin C and resveratrol were shown to be almost equal against tyrosinase and melanin biosynthesis in the cells. The IC(50) values of gnetin C activity against tyrosinase and melanin biosynthesis were 7.0 and 7.6 µM, respectively, whereas resveratrol demonstrated IC(50) values of 7.2 and 7.3 µM, respectively. These results indicated that gnetin C inhibited melanogenesis, in a manner similar to that of resveratrol, by inhibiting tyrosinase and may therefore function as a new skin-whitening agent. However, the direct effects of gnetin C and resveratrol on murine tyrosinase activities were not equal. The IC(50) value of resveratrol was 10.1 µM, while gnetin C only exhibited a 25.2% enzyme inhibition at 16 µM. The IC(25) values for gnetin C and resveratrol were 15.5 and 4.0 µM, respectively. Therefore, it is suggested that the effects of gnetin C may be due to mechanisms other than the direct inhibition of tyrosinase activity.

Aims Although increased Na+/Ca2+ exchanger 1 (NCX1) expression is observed during heart failure (HF), the pathological role of NCX1 during the progression of HF remains unclear. We examined alterations of NCX1 expression and activity in hearts after transverse aortic constriction (TAC) surgery and explored whether NCX1 influences pressure overload-induced pathological cardiac remodelling. Methods and results We generated novel transgenic mice in which NCX1 expression is controlled by a cardiac-specific, doxycycline (DOX)-dependent promoter. In the absence of DOX, TAC surgery caused substantial chamber dilation with a gradual decrease in contractility by 16 weeks. Cardiomyocytes showed a decline in contractility with abnormal Ca2+ handling during excitation–contraction (E–C) coupling. Reduced NCX1 activity was observed 8 weeks after TAC and was still apparent at 17 weeks. Induced NCX1 overexpression by DOX treatment starting 8 weeks after TAC returned NCX1 activity to pre-TAC levels and prevented chamber dilation with cardiac dysfunction. DOX treatment not only upregulated NCX1 expression in TAC-operated hearts but also returned L-type Ca2+ channel and sarcoplasmic reticulum (SR) Ca2+ ATPase expression levels to those in sham-operated hearts. In DOX-treated myocytes, contractility, T-tubule integrity, synchrony of Ca2+ release from the SR, and Ca2+ handling during E–C coupling was preserved 16 weeks after TAC surgery. In addition, DOX treatment attenuated the down-regulation of survival signalling and up-regulation of apoptosis signalling 16 weeks after TAC surgery. Conclusion Induced overexpression of NCX1 attenuated pressure overload-induced pathological cardiac remodelling. Thus, maintaining NCX1 activity may be a potential therapeutic strategy for preventing the progression of HF.

In response to atherogenic stimuli, blood monocytes transmigrate across the vascular endothelium not only through endothelial cell-cell junctions (para-cellular) but also through endothelial cells themselves (trans-cellular). The molecular mechanism of the latter is mostly unknown, because it rarely happens, especially in vitro. Although many reports have recognized trans-cellular migration from snapshot images of leukocytes halfway across the endothelium at non-junctional locations, it often produces a false-positive result, because some leukocytes that initiate trans-cellular migration withdraw and return to the apical endothelial surface. Thus, analyzing the entire process is essential. In this study, complete monocyte trans-cellular migration was successfully captured for live cells, with simultaneous visualization of endothelial PECAM-1. We suggest the possible existence of both PECAM-1-related migration at peri-junctional sites and PECAM-1-unrelated migration at sites remote from junctions. This is the first report to describe the entire process of monocyte trans-cellular migration for live cells and its relationship with endothelial PECAM-1.

福島第一原子力発電所事故後36日目(4月20日)に福島県の水田及び畑作土壌を採取し放射性のセシウム並びにヨウ素の溶出実験を行った。4月20日に採取した土壌には137Cs，134Cs及び131Iが検出され，水を用いた場合の溶出率は水田，畑土壌共に，約20％の137Cs，134Cs及び131Iが溶出した。抽出を繰り返しても2回目以降の溶離はほとんどみられなかった。水田土壌については，ヨウ化カリウム，ヨウ化セシウム，肥料，消石灰或いはセメント等を加えて1日放置して溶離を試みたが，溶出率は水抽出と同様の結果であった。

Concentration profiles of {sup 134}Cs and {sup 137}Cs were obtained in an undisturbed paddy field before plowing in Fukushima prefecture on May 24th, 2011, about two month after explosions of Fukushima Daiichi nuclear power plant. Top soil of 0 - 15cm was sampled, by separating it into 6 layers of 1 - 5cm thick. Resulting concentration profile indicates that 88% of {sup 134}Cs+{sup 137}Cs existed within 0 - 3cm and 96% within 0 - 5cm layer. However, although most of radioactive Cs stays within a few cm surface layer, a part of it already reached 10 - 15cm layer. Mean travel distance of {sup 134}Cs and {sup 137}Cs were calculated from the profile to be 1.74cm; on the other hand, mean travel distance of water molecules during the 70days was also estimated as [precipitation - evaporation]/[volumetric water content] to be about 20cm. Therefore, convective velocity of Cs was 1/10 of water due to sorption of Cs on soil. However, this ratio of Cs velocity is 2 - 3 order greater than that calculated from soil-soil solution distribution coefficients (K{sub d}) for soils of Japanese paddy field shown in a literature, indicating that the movement of Cs in the field occurs in condition far from sorption equilibrium. (author)

福島第一原子力発電所事故の3週後(4月8日)及び12週後(6月3日)に東京大学大学院農学生命科学研究科附属牧場(原発から直線距離で約130km南方の茨城県笠間市に位置する)において採取した土壌，牧草，乳牛飲料用井戸水，及び牛乳中の131I，134Cs及び137Csの放射能濃度を測定したので報告する。測定の結果，飼料中の放射性核種は速やかに牛乳中に移行することがわかった。しかし，いずれの測定でも牛乳中の131I，134Cs及び137Csの濃度は国の暫定許容値(放射性ヨウ素：300Bq/kg，放射性セシウム：200Bq/kg)を下回る値であった。

著者らは，2011年3月11日の東日本大震災に伴う福島第一原子力発電所事故由来の放射性物質の麦への蓄積様式について，γ線放出核種の同定及び分布について明らかにした。5月15日のコムギについて核種分析したところ，134Csと137Csが検出され，これらを足した放射性セシウム濃度は，枯葉で約284500Bq/kgと穂の約300Bq/kgと比較して約1000倍と突出して高い値であった。次に，5月26日のコムギについて，各葉位，穂及び茎に分けて同様に測定したところ，放射性セシウム濃度は，事故当時既に展開していた葉において高く，事故後展開した葉も含め，古い葉の順に高い値であり，穂が最も低い濃度であった。これら放射性物質の分布を可視化したところ，既に展開中の葉においてスポット状に強いシグナルが観察された。これらの結果から，事故時展開していた葉で高濃度に検出される放射性物質は，放射性降下物が直接付着したものが主であることが示唆された。一方で，事故時展開していなかった葉においても，古い順に放射性セシウム濃度が高かったことから，植物体内において葉へ移行した放射性セシウムは転流(再分配)されにくいことが示唆された。

The cationic gold(I)-catalyzed reaction of 1-alkynyl-2-allylsilylbenzenes with water results in intramolecular allylation of the alkynes via 7-exo-dig cyclization to give 1,4-dienes in good yield with excellent stereoselectivities.

Background Idiopathic pulmonary arterial hypertension (IPAH) is a disease characterized by progressively increased resistance of pulmonary arteries. In this study, we evaluated the mechanical property of single pulmonary artery smooth muscles cells (PASMC) from patients with IPAH and tested whether the PASMC showed abnormal response to a vasodilator by use of an atomic force microscope (AFM). Methods PASMC were isolated and cultured from explanted lungs of 7 patients with IPAH (IPAH-PASMC). Normal vascular specimens from 3 patients with bronchogenic carcinoma were used as normal controls (normal PASMC). The nano/micro-order elasticity of five to ten living PASMC in each sample was measured by parabolic force curves of cantilever deflection/indentation obtained by using an AFM. The elasticity measurements were performed under control conditions and under condition of nitric oxide (NO) treatment (190 and 380 nmol/L). Results There was no significant difference between nano/micro-order elasticity of normal PASMC and that of IPAH-PASMC under the control conditions. In normal PASMC, NO (190 and 380 nmol/L) significantly reduced (i.e., softened) the nano/micro-order elasticity. However, NO did not reduce elasticity in IPAH-PASMC, indicating higher vasodilator-resistive nano/micro-order rigidity in IPAH-PASMC. Conclusion Nano/micro-order elasticity change in PASMC in response to vasodilation induced by NO is reduced in patients with IPAH.

Monocyte accumulation in the arterial intima is a hallmark in early atherosclerosis. Monocyte extravasation involves sequential processes including rolling, adhesion, and transmigration across vascular endothelial cells (ECs), where abundant mechanical interactions between these two cells exist. Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a junctional protein expressed on both cells, and participates in paracellular transmigration via homophilic binding between these cells, while PECAM-1 binds homophilically between neighboring ECs in the absence of monocytes. During monocyte transmigration, PECAM-1-bearing membrane can be recruited to the transmigration spot from intracellular pools. The mechanism by which PECAM-1 binding between ECs in control state is switched to that between monocytes and ECs during transmigration, and its relationship with recruited PECAM-1 remain unclear. In this study, we built an experimental model in vitro for studying molecular behavior of PECAM-1 during monocyte transmigration. A plasmid vector containing PECAM-1 tagged with green (GFP) or red (DsRed) fluorescent protein was constructed, and separately transfected into ECs. The mixture of both transfectants in culture achieved a monolayer that contains an intercellular PECAM-1 boundary between PECAM-1-GFP- and PECAM-1-DsRed-expressing cells which is visualized as yellow in merged image. Using this model together with confocal laser scanning microscope-based system, molecular behavior of PECAM-1 on neighboring ECs during monocyte transmigration was observed in live cells. This model can be essential to directly visualize binding/dissociation state of PECAM-1 between neighboring ECs during monocyte trans-endothelial migration in relation to mechanical monocyte-EC interactions.

The effects of Brazilian green propolis ethanol extract on Cry j1-induced cys-leukotrienes and histamine release from peripheral leukocytes of patients with allergic rhinitis were investigated. One of the key mechanisms for the anti-allergic properties of the extract was revealed to be the suppression of cys-LTs release. Furthermore, a series of propolis components and their phenethyl esters were synthesized and evaluated as inhibitors of cys-LTs release. Artepillin C, baccharin, and kaempferide were the major active components of the ethanol extract. The inhibitory activity of artepillin C phenethyl ester was comparable to that of existing LT synthesis inhibitors.

There are mainly three types of propolis whose major anticancer ingredients are entirely different: (1) CAPE (caffeic acid phenethyl ester)-based propolis in Europe, Far East and New Zealand, (2) artepillin C (ARC)-based Brazilian green propolis and (3) Brazilian red propolis. It was shown previously that NF (neurofibromatosis)-associated tumors require the kinase PAK1 for their growth, and CAPE-based propolis extracts such as Bio 30 suppress completely the growth of NF tumors in vivo by blocking PAK1 signaling. Also it was demonstrated that ARC suppresses angiogenesis, suggesting the possibility that ARC also blocks oncogenic PAK1 signaling. Here it is shown for the first time that both ARC and green propolis extract (GPE) indeed block the PAK1 signaling selectively, without affecting another kinase known as AKT. Furthermore, it was confirmed that ARC as well as GPE suppress almost completely the growth of human NF tumor xenografts in mice, as does Bio 30. These results suggest that both CAPE-based and ARC-based propolis extracts are natural anti-PAK1 remedies and could be among the first effective NF therapeutics available on the market. Since more than 70% of human cancers such as breast and prostate cancers require the kinase PAK1 for their growth, it is quite possible that GPE could be potentially useful for the treatment of these cancers, as is Bio 30.

Angiotensin I-converting enzyme (ACE) inhibitory and hypotensive effects of 7 peptide fractions (Frs) of royal jelly protein hydrolysate (RJPH) were studied in comparison with those of RJPH alone. Fr 4 and Fr 5 were the highest in ACE inhibitory activity and yield, respectively. Molecular weights (MWs) of RJPH and Fr 1-Fr 7 were distributed from 100 to 5,000 and those of Fr 1-Fr 7 increased in order from Fr 1 to Fr 7. RJPH, Fr 3 and Fr 4 at doses of 10, 30 and 100mg/kg i.v. and Fr 5 and Fr 6 at doses of 30 and 100mg/kg i.v. caused transiently significant hypotensive effects in spontaneously hypertensive rats (SHR). Fr 3, Fr 4, Fr 5 and Fr 6 at a dose of 1,000mg/kg also caused significant hypotensive effects 3h, 4-5h, 7-8h and 8h after oral administration in SHR, respectively. RJPH caused a long-lasting hypotensive effect in proportion to the magnitude of the MWs of RJPH fractions. The hypotensive pattern of RJPH was similar to the combined pattern of Fr 3-Fr 6. From these results, it can be concluded that the long-lasting hypotensive effect of oral administration of RJPH is dependent on the MWs of its ACE inhibitory peptides and the time required to digest them.

Two new spirostanol glycosides (1, 2) and a new furostanol glycoside (3), together with nine known steroidal glycosides (4-12) were isolated from the leaves of Furcraea foetida (Agavaceae). The structures of the new compounds were determined by spectroscopic analysis and the results of hydrolytic cleavage. The isolated compounds were evaluated for their cytotoxic activities against HL-60 human leukemia cells, A549 human lung adenocarcinoma cells, HSC-2 human oral squamous carcinoma cells, and HSC-4 human oral squamous carcinoma cells.

Three new triterpene glycosides (1-3), together with eight known triterpene glycosides (4-11), were isolated from the whole plant of Anemone hupehensis var. japonica (Ranunculaceae). The structures of the new compounds were determined on the basis of spectroscopic analysis and the results of hydrolytic cleavage experiments. The isolated compounds were evaluated for their cytotoxic activities against HL-60 human leukemia cells, HSC-2 human oral squamous carcinoma cells, HSC-4 human oral squamous carcinoma cells, and A549 human lung adenocarcinoma cells.

Osteocytes acquire their stellate shape during the process of changing from osteoblasts in bone. Throughout this process, dynamic cytoskeletal changes occur. In general, changes of the cytoskeleton affect cellular mechanical properties. Mechanical properties of living cells are connected with their biological functions and physiological processes. In this study, we for the first time analyzed elastic modulus, a mechanical property of bone cells. Bone cells in embryonic chick calvariae and in isolated culture were identified using fluorescently labeled phalloidin and OB7.3, a chick osteocyte-specific monoclonal antibody, and then observed by confocal laser scanning microscopy. The elastic modulus of living cells was analyzed with atomic force microscopy. To examine the consequences of focal adhesion formation on the elastic modulus, cells were pretreated with GRGDS and GRGES, and then the elastic modulus of the cells was analyzed. Focal adhesions in the cells were visualized by immunofluorescence of vinculin. From fluorescence images, we could distinguish osteoblasts, osteoid osteocytes and mature osteocytes both in vivo and in vitro. The elastic modulus of peripheral regions of cells in all three populations was significantly higher than in their nuclear regions. The elastic modulus of the peripheral region of osteoblasts was 12053 ± 934 Pa, that of osteoid osteocytes was 7971 ± 422 Pa and that of mature osteocytes was 4471 ± 198 Pa. These results suggest that the level of elastic modulus of bone cells was proportional to the stage of changing from osteoblasts to osteocytes. The focal adhesion area of osteoblasts was significantly higher than that of osteocytes. The focal adhesion area of osteoblasts was decreased after treatment with GRGDS, however, that of osteocytes was not. The elastic modulus of osteoblasts and osteoid osteocytes were decreased after treatment with GRGDS. However, that of mature osteocytes was not changed. There were dynamic changes in the mechanical property of elastic modulus and in focal adhesions of bone cells.

We investigated whether artepillin C, a major component of Brazilian propolis, acts as a neurotrophic-like factor in rat PC12m3 cells, in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of PC12m3 cells were treated with artepillin C at a concentration of 20 microM, the frequency of neurite outgrowth induced by artepillin C was approximately 7-fold greater than that induced by NGF alone. Artepillin C induced-neurite outgrowth of PC12m3 cells was inhibited by the ERK inhibitor U0126 and by the p38 MAPK inhibitor SB203580. Although artepillin C-induced p38 MAPK activity was detected in PC12m3 cells, phosphorylation of ERK induced by artepillin C was not observed. On the other hand, artepillin C caused rapid activation of ERK and the time course of the activation was similar to that induced by NGF treatment in PC12 parental cells. However, NGF-induced neurite outgrowth was inhibited by artepillin C treatment. Interestingly, inhibition of ERK by U0126 completely prevented artepillin C-induced p38 MAPK phosphorylation of PC12m3 cells. These findings suggest that artepillin C-induced activation of p38 MAPK through the ERK signaling pathway is responsible for the neurite outgrowth of PC12m3 cells.

In a previous study (Y. Kimura et al., Biosci. Biotechnol Biochem., 70, 2583-2587, 2006), we found that new complex type N-glycans harboring Thomsen-Friedenreich antigen (Ga1 beta 1-3GalNAc) unit occur on royal jelly glycoproteins, suggesting the involvement of a new beta 1-3galactosyltransferase in the synthesis of the unusual complex type N-glycans. So far, such beta 1-3galactosyltransferase activity, which can transfer galactosyl residues with the beta 1-3 linkage to beta 1-4 GalNAc residues in N-glycan, has not been found among any eucaryotic cells. But using GalNAc(2)GlcNAc(2)Man(3)-GlcNAc(2)-PA as acceptor N-glycan, we detected the beta 1-3 galactosyltransferase activity in membrane fraction prepared from honeybee cephalic portions. This result indicates that honeybee expresses a unique beta 1-3 galactosyltransferase involved in biosynthesis of the unusual N-glycan containing a tumor related antigen in the hypopharyngeal gland.

The effect of bee pollen cistus extract on serum and bone biochemical components in streptozotocin (STZ)diabetic rats was investigated. The water-solubilized extracts were obtained from the bee pollen of Cistus ladaniferus .R ats received a single subcutaneous administration of STZ (6.0 mg/100 g body weight), and then the animals were orally administered water-solubilized extract (5, 10, or 20 mg/100 g body weight) of bee pollen cistus once daily for 14 days. The administration of STZ caused a significant decrease in body weight and a significant increase in serum glucose, triglyceride, and calcium levels, indicating a diabetic state. These alterations were significantly prevented by the administration of the extract (5, 10, or 20 mg/100 g). Serum inorganic phosphorus concentration was significantly decreased in STZ-diabetic rats, and the decrease was significantly prevented after administration of the extract of 10 or 20 mg/100 g. Calcium content and alkaline phosphatase activity in the femoral-diaphyseal and -metaphyseal tissues were significantly decreased in STZ-diabetic rats. These decrease were significantly prevented after administration of the extract of 10 or 20 mg/100 g. The disphyseal DNA content was also significantly decreased in STZ-diabetic rats. This decrease was significantly prevented after the administration of the extract of 10 or 20 mg/100 g. This study demonstrates that the intake of bee pollen cistus extract has preventive effects on bone loss in STZ-diabetic rats, and that the intake has partial restorative effects on serum biochemical findings with the diabetic state.

Four phenolic compounds were isolated from Japanese butterbur (Petasites japonicus) by preparative RP-HPLC technique, and were identified as chlorogenic acid (CGA), fukinolic acid (FA), 3,5-dicaffeoyl quinic acid, and 3,4,5-tricaffeoyl quinic acid. Since the antioxidant activity of FA among these four compounds has not been reported in detail, we investigated the scavenging activity of FA against several active oxygens and radicals, compared with CGA, gallic acid (GA) and epigallocatechin gallate (EGCG), using ESR spectroscopy. The order of potency of scavenging superoxide anion, nitric oxide (NO) and DPPH was EGCG>FA>CGA. The order of potency of scavenging hydroxyl radical was EGCG>FA>GA. FA inhibited the lipopolysaccharide-stimulated NO production and inducible NO synthase protein and mRNA expression by mouse macrophage-like cells Raw 264.7 more potently than CGA. The present study suggests the usefulness of FA as an antioxidant.

Melinjo (Gnetum gnemon L.) seed extracts (MSEs) are rich in resveratrol dimers (gnemonoside A, C, D, gnetin C), trans-resveratrol, and other resveratrol derivatives. trans-Resveratrol is a widely studied caloric restriction mimetic. In mice fed a high-fat diet (HFD), trans-resveratrol protects against obesity, type 2 diabetes, and premature death. Here, treatment of HFD-fed mice with 2.0% MSE significantly reduced body weight gain (p

On March 11, 2011, Japan's northeast Pacific coast was hit by a gigantic earthquake and subsequent tsunami. Soma City in Fukushima Prefecture is situated approximately 44 km north of Fukushima Daiichi Nuclear Power Plant. Soma General Hospital is the only hospital in Soma City that provides full-time otolaryngological medical care. We investigated the changes in new patients from one year before to three years after the disaster. We investigated 18,167 new patients treated at our department during the four years from April 1, 2010 to March 31, 2014. Of the new patients, we categorized the diagnoses into Meniere's disease, acute low-tone sensorineural hearing loss, vertigo, sudden deafness, tinnitus, and facial palsy as neuro-otologic symptoms. We also investigated the changes in the numbers of patients whom we examined at that time concerning other otolaryngological disorders, including epistaxis, infectious diseases of the laryngopharynx, and allergic rhinitis. The total number of new patients did not change remarkably on a year-to-year basis. Conversely, cases of vertigo, Meniere's disease, and acute low-tone sensorineural hearing loss increased in number immediately after the disaster, reaching a plateau in the second year and slightly decreasing in the third year. Specifically, 4.8% of patients suffering from these neuro-otologic diseases had complications from depression and other mental diseases. With regard to new patients in our department, there was no apparent increase in the number of patients suffering from diseases other than neuro-otologic diseases, including epistaxis, and allergic rhinitis. Patients suffering from vertigo and/or dizziness increased during the first few years after the disaster. These results are attributed to the continuing stress and tension of the inhabitants. This investigation of those living in the disaster area highlights the need for long-term support.

Background External auditory canal cholesteatoma (EACC) is a rare otologic disease, characterized by focal osteonecrosis, sequestration and overlying epithelial loss of the bony external auditory canal (EAC). The etiology and pathogenesis of EACC remain controversial. There are only 2 reports on the association between EACC and chronic renal failure (CRF)/hemodialysis (HD). METHOD & RESULT: (1) This study reviewed seven EACC cases with CRF. The mean age was 68.4 years (range: 56 -81 years), and the male-female ratio was 5:2. There were 12 ears with EACC (5 cases were bilateral and 2 cases were unilateral). The EACCs were found in the inferior or posterior inferior part of the EAC in 11 ears. The number of the ears in stage III or IV was 6. Five cases were on HD. (2) Seventy-six cases with CRF on HD were examined for EACC, and 2 out of those 70 cases were diagnosed as having EACC. (3) A comparative study of the 7 EACC cases on HD and 68 non-EACC cases on HD revealed no significant differences in the sex, age, period on CRF/HD, complications (diabetes mellitus or skin disease), smoking, ear cleaning and the use of an earphone or a hearing aid. Conclusion & discussion Six out of all 9 EACC cases with CRF developed bilaterally, and in the inferior or posterior inferior part of the EAC, which implies a common pathological condition that contributes to the development of EACC. The mean age of 9 patients with EACC was relatively older (66.7 years), therefore age-related changes in the EAC are suspected in the cases with CRF on HD. The patients with CRF on HD have a high incidence of EACC. This strongly suggests the association between EACC and CRF/HD, but the mechanism of this pathogenesis has not been revealed.

Purpose To compare the mechanical properties of sliding lengthening (SL) and Z-lengthening (ZL) for flexor tendon elongation used for conditions such as Volkmann contracture, cerebral palsy, and poststroke spasticity. Methods We harvested 56 flexor tendons, including flexor pollicis longus tendons, flexor digitorum superficialis tendons (zones II to IV), and flexor digitorum profundus tendons (zones II to V) from 24 upper limbs of 12 fresh cadavers. Each tendon was harvested together with its homonymous tendon from the opposite side of the cadaver and paired. We used 28 pairs of tendons and divided them randomly into 4 groups depending on the lengthening distance (20 or 30 mm) and type of stitching (single or double mattress sutures). Then we divided each pair into either the SL or ZL group. Each group was composed of 7 specimens. The same surgeon lengthened all tendons and stitched them with 2-0 polyester sutures. We tested biomechanical tensile strength immediately after completing lengthening and suturing in each group. Results Ultimate tensile strengths were: 23 N for the SL 20-mm lengthening and single mattress suture and 7 N for the ZL; 25 N for the SL 20-mm lengthening and double mattress suture and 10 N for the ZL; 15 N for the SL 30-mm lengthening and single mattress suture and 8 N for the ZL; and 18 N for the SL 30-mm lengthening and double mattress suture and 10 N for the ZL. Conclusions The SL technique may be a good alternative to the ZL technique because it provides higher ultimate tensile strength. Clinical relevance Because of its higher ultimate tensile strength, the SL technique may allow for earlier rehabilitation and reduced risk of postoperative complications.

We noticed intracytoplasmic membranous, annular, or circular structures in the lesion of pemphigus foliaceus and studied these by regular transmission electron microscopy and immunoelectron microscopy. These curvicircular bodies were observed in the preacantholytic keratinocytes of the blister wall as well as in acantholytic cells in 6 out of 6 patients with pemphigus foliaceus. They were absent in samples from 3 patients with pemphigus vulgaris. These structures were about 60-70 nm wide and consisted of 4 electron-dense layers. They were continuous with intact desmosomal structures and gap junctions in the periphery of the keratinocytes. These curvicircular membranous bodies were well labeled with immunogold particles for desmoglein, plakoglobin, connexin 43, and IgG. In contrast to pemphigus vulgaris, splitting of desmosomes through dissolution of intercellular desmoglea was seldom observed in all 6 specimens of pemphigus foliaceus. These findings suggest that in pemphigus foliaceus 1) curvicircular bodies are derived from internalized desmosomes and gap junctions, and 2) cell-to-cell adhesions are weakened by this internalization and acantholysis is initiated, while in pemphigus vulgaris the dissolution of desmoglea is the initial event. It is suggested that in pemphigus foliaceus the binding of autoantibody induces internalization of many intact desmosomes and gap junctions rather than splitting them.

Six-stroke diesel engine proposed here has six processes in one cycle, i.e. intaka, 1st compression, 1st combustion, 2nd compression, 2nd combustion and exhaust. By the effect of direct EGR in the 2nd combustion process, NO concentration could be expected to decrease. However, by a weak mixing in the 2nd combustion process, much soot was exhausted and NO concentration was hardly decreased compared with the conventional four-stroke diesel engine. To improve the fuel/air mixing at the 2 nd combustion process, high pressure injection with common rail system was applied to the engine. As the result, NO concentration was increased owing to the increase of premixed combustion of 1st combustion process, although soot was reduced. Therefore, the 1st injection timing was extremely advanced to control the 1st combustion process. It changed 1st combustion process into HCCI-like combustion, and NO concentration was greatly decreased. Moreover, soot was reduced with no increase of NO concentration in the 2nd combustion process. As the result, trade-off curve of NO and soot was improved compared with the conventional four-stroke diesel engine. But CO concentration was increased.

The effects of bee pollen extract on bone components in the femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues of rats in vitro and in vivo were investigated. Bone tissues were cultured for 48 hr in serum-free Dulbecco's modified Eagle's medium containing either vehicle or water- or ethanol-solubilized extracts (10, 100, or 1000 μg/ml of medium) obtained from the bee pollen of Cistus ladaniferus. Calcium content in the femoral-diaphyseal or -metaphyseal tissues was significantly increased in the presence of water-solubilized extract (100 or 1000 μg/ml) and ethanol-solubilized extract (1000 μg/ml). An increase was also observed in the presence of water-solubilized extract (100 μg/ml) obtained from Fagopyrum esculentum, Camellia sinesis, or Brassica napus L. Alkaline phosphatase activity and DNA content in the femoral-diaphyseal or -metaphyseal tissues in vitro were significantly increased in the presence of water-solubilized extract (100 or 1000 μg/ml) obtained from the bee pollen. The effects of the bee pollen extract (100 μg/ml) in increasing bone components were completely inhibited in the presence of cycloheximide (10-6 M), an inhibitor of protein synthesis, in vitro. Moreover, the calcium content and alkaline phosphatase activity in the femoral-diaphyseal or -metaphyseal tissues were significantly increased by the oral administration of water-solubilized extracts (5 or 10 mg/100 g body weight) obtained from the bee pollen of Cistus ladaniferus once daily for 7 days. The DNA content in the diaphyseal or metaphyseal tissues was significantly increased by the oral administration of water-solubilized extract (10 mg/100 g) of bee pollen cistus. The dose of 1.0 mg/100 g caused a significant increase in the diaphyseal and metaphyseal alkaline phosphatase activity or the metaphyseal DNA content in vivo. This study demonstrates that the extract of bee pollen has an anabolic effect on bone components in rats in vitro and in vivo.

Royal jelly (RJ) has been used worldwide for many years as medical products, health foods and cosmetics. Since RJ contains testosterone and has steroid hormone-type activities, we hypothesized that it may have beneficial effects on osteoporosis. We used both an ovariectomized rat model and a tissue culture model. Rats were divided into eight groups as follows: sham-operated (Sham), ovariectomized (OVX), OVX given 0.5% (w/w) raw RJ, OVX given 2.0% (w/w) RJ, OVX given 0.5% (w/w) protease-treated RJ (pRJ), OVX given 2.0% (w/w) pRJ, OVX given 17β-estradiol and OVX given its vehicle, respectively. The Ovariectomy decreased tibial bone mineral density (BMD) by 24%. Administration of 17β-estradiol to OVX rats recovered the tibial BMD decrease by 100%. Administration of 2.0% (w/w) RJ and 0.5–2.0% (w/w) pRJ to OVX rats recovered it by 85% or more. These results indicate that both RJ and pRJ are almost as effective as 17β-estradiol in preventing the development of bone loss induced by ovariectomy in rats. In tissue culture models, both RJ and pRJ increased calcium contents in femoral-diaphyseal and femoral-metaphyseal tissue cultures obtained from normal male rats. However, in a mouse marrow culture model, they neither inhibited the parathyroid hormone (PTH)-induced calcium loss nor affected the formation of osteoclast-like cells induced by PTH in mouse marrow culture system. Therefore, our results suggest that both RJ and pRJ may prevent osteoporosis by enhancing intestinal calcium absorption, but not by directly antagonizing the action of PTH.