| 1.
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Itabashi, Yukihiro ; Baba, Toshiaki ; Kato, Satoru ; Hakamada, Kenichi
概要:
Forty patients with an average age of 64.6 years （range: 45-83） with early gastric cancer of cT1 （M,SM） underwent a cur
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ative-intended laparoscopy-assisted distal gastrectomy （LADG） where laparoscopic D1+αnodal clearance and extra-abdominal Billroth-I stapled anastomosis were performed. There was no conversionto open gastrectomy. The operation time for the 40 cases ranged 150 - 482 min （median: 285）, while that for thelatest 10 cases reduced to 154-278 min （median: 216）. The length of hospital stay of the patients varied 10-85 days（median: 17）. Postoperative complications encountered were anastomosis-related: 2 anastomotic bleeding and 2anastomotic passage disturbance（ 1 stricture and 1 temporary stenosis） occurred but no dehiscence. Four inaccuratepreoperative diagnoses of tumor invasion depth were revealed by postoperative pathology of the resected specimens.Thereafter the accuracy in the preoperative diagnosis was highly enhanced with implementation of endoscopicultrasound. Recurrence occurred in one patient with pT2（ SS） pN2, who died of pleural carcinomatosis 4 years and 3months after surgery. As the reduced operation time in LADG came closer to that in open distal gastrectomy, we will continuethis procedure for early gastric cancer. For this, the importance of an accurate preoperative diagnosis can’t beoveremphasized.
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| 2.
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Yoshida, Ikko ; Ishizaka, Hiroshi ; Hasegawa, Kazushi ; Satoh, Kiyohiko ; Osanai, Tomohiro ; Motomura, Shigeru ; Okumura, Ken
概要:
Objectives The purpose of this study was to test the hypothesis that adenosine-induced coronarymicrovascular dilation is
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blunted in the animals with diabetes mellitus（ DM） through the impairment of KATP channelfunction. Background Adenosine-induced coronary vasodilation is demonstrated to be mediated by activation of ATPsensitivepotassium（ KATP） channels and nitric oxide（ NO）. Methods The hearts of Otsuka Long-Evans Tokushima fatty rats （OLETF, type 2 DM rats）, and control Long-Evans Tokushima fatty rats（ LETO） at the ages of 32 and 8 weeks were perfused using a Langendorff system withconstant perfusion pressure （80 mmHg）. Changes in coronary fl ow to adenosine, pinacidil and sodium nitroprusside（SNP） were examined before and after administration of glibenclamide（ 10-7 M）, or NG-nitro-L-arginine methyl ester（L-NAME, 10-4 M）. Results At the age of 32 weeks, adenosine- and pinacidil-induced increases in coronary fl ow were blunted in OLETFas compared with those in LETO （both p<0.05）. Glibenclamide attenuated adenosine-induced increase in coronaryfl ow in LETO （p<0.05）, but not in OLETF. In contrast, L-NAME attenuated adenosine-induced increase in coronaryflow in OLETF （p<0.05）, but not in LETO. SNP-induced increases in coronary flow in LETO and OLETF werecomparable and were not aff ected by glibenclamide. In 8-week-old OLETF and LETO, no diff erence was observed inadenosine-, pinacidil- and SNP-induced increases in coronary fl ow between OLETF and LETO. Conclusions In this type 2 DM model, KATP channel function in coronary microcirculation is impaired. Adenosineinducedincrease in coronary fl ow is mediated mainly by NO mechanism.
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| 3.
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Sutoh, Takemichi ; Fukuda, Ikuo ; Kimura, Daisuke ; Tsushima, Takao
概要:
Nafamostat mesilate（ FUT-175）, a synthetic serine protease inhibitor, has been reported to have antitumouractivities tow
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ard solid tumours. The objective of this study was to characterize the biological activities of FUT-175 ina malignant mesothelioma cell line. We used MSTO-211H, a biphasic-type human malignant pleural mesothelioma cellline. The effect on cell growth was evaluated by 3（- 4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay.Secretion of urokinase-type plasminogen activator（ u-PA） and plasminogen activator inhibitor-1（ PAI-1） was analysedby enzyme-linked immunosorbent assay. The eff ects on relative mRNA expression levels were measured by reversetranscription polymerase chain reaction. The eff ect on cell invasiveness was evaluated by cell invasion assay. FUT-175at 10-5 M signifi cantly inhibited cell growth and cell invasiveness. Cell growth reduced to 47.0 ± 2.1% compared withthe control. The number of invasive cells also reduced to 16.0 ± 0.7 cells/hpf, while that of control cells was 41.4 ± 8.0cells /hpf. U-PA and PAI-1 secreted from the cells were also reduced by FUT-175 in a dose-dependent manner. Theseresults suggest that FUT-175 has the potential to act as a therapeutic agent against local growth and invasion, andfunctions by reducing PAI-1 and u-PA production of the human malignant mesothelioma（195 words）
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| 4.
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Nodagashira, Tatsuya ; Odagiri, Hiroki ; Ikenaga, Shojiro-Kazunori ; Maruyama, Masateru ; Sato, Toshiyuki ; Hakamada, Kenichi
概要:
Tissue-specifi c promoter has been used for cancer-specifi c suicide gene therapy, but its transcriptionalactivity is r
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elatively low. For more effi cient gene therapy of HER2-expressing tumor, a double adenovirus infectionsystem was established, in which a ‘regulator’ vector carried Cre gene under the control of HER2 promoter and ‘target’vectors carried target genes activated by Cre. We constructed a Cre recombinase expression vector, AxHER2Cre, forthe ‘regulator’ vector. By the combination of this vector and AxCALNLZ, β-D-galactosidase was induced in 90% and70% of MKN7 and MDA-MB-453, HER2‒overexpressing cell lines, but only about 20% and 10% of MKN28 and MCF7,low HER2-expressing cell lines. By the quantifi cation analysis, the β-galactosidase activities induced by this systemwere comparable to those by the combination of AxCANCre and AxCALNLZ. These results indicated that Cre/loxP system under the regulation of HER2 promoter could induce effi cient gene expression, maintaining the HER2-expression specifi city. Breast cancer with HER2 overexpression is treated with trastuzumab. However, refractoryor resitance of HER2 positive breast cancer against trastuzumab becomes a severe clinical problem, recently. Thissystem seemed to be another therapeutic option.
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| 5.
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Sato, Hideaki ; Sato, Masashi ; Chiba, Makie ; Ito, Kyoko ; Ito, Koichi
概要:
The ability of murine allogeneic umbilical cord blood cells （UCBCs） to reconstitute the immune systemwas investigated. U
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CBCs obtained from fetuses of C57BL/6 （B6; H-2b） mice, which were transgenic for greenfluorescent protein （GFP）, were transplanted into RAG2 （-/-） BALB/c mice （H-2d）. After transplantation, flowcytometric analysis revealed successful reconstitution of phenotypically mature GFP-positive immune cells of donororigin, including T cells, B cells, monocytes, and granulocytes in the peripheral blood of the recipient mice. Analysisof functional maturation of lymphocytes revealed that 2,4,6-trinitrophenyl-keyhole limpet hemocyanin （TNP-KLH）-immunized UCBC-transplanted recipients produced both TNP-specific IgM and IgG antibodies. These resultsindicated that the recipient mice were capable of mounting antibody responses to T-dependent antigens; further, Igclass switching from IgM to IgG confi rmed that both B cells and CD4+ helper T cells derived from allogeneic UCBCswere immunologically competent. Furthermore, mice transplanted with allogeneic UCBCs accepted skin grafts fromboth B6 and BALB/c mice. However, these chimeric mice completely rejected skin grafts from third party C3H/HeJ （H-2k） mice, indicating the presence of functional CD8+ killer T cells as well as CD4+ helper T cells. In termsof potential clinical application, our results indicate that allogeneic UCBC transplantation can enable recovery of thenormal immune system in recipients.
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| 6.
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Ito, Kyoko ; Masuko, Kazutaka ; Ito, Koichi
概要:
Contact between dendritic cells（ DCs） and resting T cells is essential for initiation of the primary immuneresponse. In
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this study, we examined whether neuronal leucine-rich repeat protein 3 （NLRR3）, a receptor involvedin nerve system development, participates in DC-T cell binding and T-cell activation. We confirmed that NLRR3is expressed on naive T cells. NLRR3 contains an Arg-Gly-Asp （RGD） motif in its amino acid sequence, for whichseveral integrins can be considered as potential ligands. Indeed, monocyte-derived DCs expressed several integrinsthat can bind to the RGD motif. Functionally, DC-induced resting allogeneic T-cell proliferation was partially inhibitedby addition of integrin-specific antibodies and synthetic RGD peptides, indicating that RGD-containing molecules,including NLRR3 and several integrins, at least participate in the events of the initial primary immune responseinvolving naive T cells and DCs. Furthermore, DCs were shown to bind directly to NLRR3-transfected Chinesehamster ovary cells in a NLRR3-dependent manner, and the binding was removed in the presence of integrin-specifi cantibodies. These data suggest that NLRR3 plays an important role in initiation of the primary immune response.
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| 7.
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Yokoyama, Hiroaki ; Saito, Shin ; Higuma, Takumi ; Hanada, Hiroyuki ; Osanai, Tomohiro ; Daitoku, Kazuyuki ; Fukuda, Ikuo ; Okumura, Ken
概要:
Adipose tissue secretes various bioactive molecules （adipokines）, and apelin is one kind of adipokines.Recently, it was
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shown that plasma apelin level is decreased in patients with chronic heart failure, and apelin mightplay an important role in the pathogenesis of cardiovascular disease. However, plasma apelin level in coronary arterydisease（ CAD） or other heart disease such as valvular heart disease（ VHD） has not been elucidated. We enrolled 31patients with CAD and 14 patients with VHD who underwent elective cardiac surgery. We also examined plasmaapelin level in 20 healthy subjects （Control）. Blood samples were obtained before the surgery. Paired samples ofvisceral and subcutaneous adipose tissues were harvested during surgery. Plasma apelin level was lower in both CADand VHD than in Control. When compared between CAD and VHD, it was lower in CAD than in VHD, and was notaff ected by treatment with HMG-CoA reductase inhibitors （statins） which was shown to increase adiponectin level.Left ventricular ejection fraction（ LVEF） was lower in CAD than in VHD. There was no correlation between plasmaapelin level and LVEF. Gene expression of apelin in visceral adipose tissue was higher than that in subcutaneousadipose tissue, but it was similar between two groups. These suggest that plasma apelin level was decreased inpatients with cardiac diseases, especially in those with CAD. Its role in the pathophysiology of CAD remains to beelucidated.
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| 8.
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Lee, Sangun ; Umeda, Takashi ; Takahashi, Ippei ; Matsuzaka, Masashi ; Danjo, Kazuma ; Iwane, Kaori ; Iwasaki, Hiroki ; Nakaji, Shigeyuki
概要:
The changes in the anthropometric and blood biochemical parameters, neutrophil functions such asreactive oxygen species
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（ROS） production capability, phagocytic activity （PA） and the profile of mood state （POMS）were measured after 2-hours of judo training session for 24 male university freshmen judoists who had not done anytraining for a long period. As for neutrophil function, PA significantly decreased after the training compared with thepre-value, though ROS production capability did not change. The Depression, Tension and total mood disturbance （TMD） decreased significantly after the training. Furthermore, the change in blood sugar correlated positively withthe changes in the Depression, Fatigue and TMD. The change in creatine kinase was positively associated with thechange in the Confusion. The training negatively acted on the appearance of physiological fatigue, and positivelyacted on the appearance of mental fatigue, and some relationship between both. Furthermore, the typical responseof the neutrophil function （PA decrease and no change in ROS） does not coincide with the typical neutrophilcompensation pattern e.g. PA decrease and ROS increase may be due to the lack of training on the part of the subjects for the last 6 months.
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| 9.
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Abe, Kazuhiro ; Higuchi, Tsuyoshi ; Mizunuma, Hideki ; Kakizaki, Ikuko ; Endo, Masahiko ; Suto, Shinichiro
概要:
The aim of study is to analyze functional sugar chains, glycosaminoglycans （GAGs） in urine frompostmenopausal women with
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osteoporosis. A 24-hour urine was collected from 15 osteoporotic women and normalwomen. All women were postmenopausal and aged at 60 to 70 years old. Diagnosis of osteoporosis was based onthe diagnosis criteria proposed by the Japan Osteoporosis Society after measurement of lumbar spine bone mineraldensity by dual energy X-ray absorptiometry. The urine was concentrated by dialysis and evaporation, thenprecipitated with ethanol. The precipitate was then reconstituted into NaCl solution, precipitated again by adding 10% cetylpyridinium chloride and the precipitation was used as complex GAGs fraction. The yield of complex fractionwas significantly higher in osteoporotic subjects than that in normal control. Then, the complex GAGs fraction wasanalyzed by cellulose acetate membrane electrophoresis and high performance liquid chromatography （HPLC）,respectively. Cellulose acetate membrane electrophoresis has revealed that GAGs in urine contained partiallydesulfated chondroitin sulfate. However, further analysis by HPLC has shown that the excretion of desulfated GAGswas rather less in osteoporotic women, indicating that urinary GAGs fraction from osteoporotic women containsunknown acidic polysaccharides as well as GAGs.
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| 10.
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大久保, 愉一 ; 松坂, 方士 ; 高橋, 一平 ; 檀上, 和真 ; 中路, 重之 ; 梅田, 孝
概要:
労働者のメンタルヘルスと社会環境学的要因を調査し，メンタルヘルスに及ぼすこれらの影響を調査した．東北，北海道の労働者6310名に家族構成，生活習慣，健康度，職場環境，抑うつ度（CES-D）をアンケートで聞き取り，CES-D と他の項目の関係
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を検討した．その結果，「仕事・職業満足度」「仕事裁量度」「仕事士気度」「上司関係満足度」「自己評価点」「健康度」「生活習慣点」「家族・友人満足度」で，低得点群に比較し高得点群でオッズ比が小さく，「悪対処数（問題となるストレス解消行動の数）」ではその逆であった．一方，「勤務時間」「時間外勤務」「仕事量質負担度」では有意な結果は得られなかった．以上より，わが国の労働者は地域社会・家庭のストレスを職場に持ち込み，さらにうつに対する感受性がうつ度に大きく影響していることが示唆された．また，仕事量質の影響は大きくなく，労働者のメンタルヘルス改善を考えた場合，社会精神文化を考慮した職場環境を構築することが重要と考えられた．
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| 11.
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川口, 英夫 ; 青木, 昌彦 ; 畑山, 佳臣 ; 小野, 修一
概要:
【目的】4D-CTを用いて呼吸性移動の線量計算に対する影響を検討した．【対象・方法】4D-CTを撮像した14例を対象に平均画像を用いて非対向6門で治療計画を作成した．計画を各呼吸位相のCTに貼り付けMU値を計算し，呼吸位相毎に平均画像のMU
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値と比較した（MUdiff ）．また，各門毎の変動を計算した（⊿MU）．呼吸性移動をビームの平面方向と垂直方向に分離し，それぞれ⊿MUとの相関を検討した．線量計算はClarkson法（C法）, superposition法（S法）の2つを用い比較した．【結果】MUdiff はC法では吸気相以外で，S法では終末吸気・呼気のみで有意差を認めた．⊿MUはC法では平面上方向・垂直方向に中等度相関を認めたが，S法では垂直方向のみに弱い相関を認めた．【結論】C法は呼吸性移動の影響を受けやすく，S法は影響が少なかった．呼吸性移動を加味した線量計算はS法が有用と考えられた．
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| 12.
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Zheng, Zhongxi ; Miura, Tomisato ; Nozaka, Hiroyuki ; Takamatsu, Terumasa ; Sato, Tatsusuke
概要:
To date, digital pathology is based on single focal plane images, and with its rapid development, image quality and information are becoming critical concern in the field. This paper presents a new multiple focal-plane based focus image
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fusion algorithm to enhance the image quality. First of all, we will introduce a new focus image fusion lgorithm; next, we will demonstrate some experimental data and results on cytopathology slide images; and finally, we will verify the effectiveness of this algorithm using computer vision techniques and cytopathology diagnosis, and draw the conclusions.
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| 13.
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Numazawa, Satomi ; Matsuzaka, Masashi ; Iwane, Kaori ; Inoue, Ryo ; Danjo, Kazuma ; Takahashi, Ippei ; Umeda, Takashi ; Nakaji, Shigeyuki
概要:
The present study investigated the relationship between obesity and atherosclerosis according to agein the general femal
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e population. Six hundred fifty-five females were divided into 3 different age groups and therelationship between brachial-ankle pulse wave velocity （baPWV） and various obesity indices were investigated.In the youngest age group （20-39 years）, BMI, percentage body fat and abdominal circumference were positivelycorrelated with baPWV. In the middle-aged group （40-59 years）, all four obesity indices （BMI, percentage bodyfat, abdominal circumference and the waist-to-hip ratio （WHR）） were positively correlated with baPWV. However,baPWV was found to be negatively correlated with BMI only in the oldest age group （60 years and over）, thoughit was still positively correlated with WHR （but not abdominal circumference）. The reason for this may be due to agradual reduction of body weigh caused by some lifestyle-related diseases or aging. In this study, it was confirmedthat abdominal obesity is a key indicator of arteriosclerosis, though the importance of BMI （general obesity） as anindicator may depend on the generation. Furthermore, our results suggested that abdominal circumference and WHRmay have different significance as the indicators of arteriosclerosis.
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| 14.
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Yamai, Kiyonori ; Umeda, Takashi ; Matsuzaka, Masashi ; Danjo, Kazuma ; Takahashi, Ippei ; Tsuya, Ryosuke ; Hasebe, Tatsuya ; Nakaji, Shigeyuki
概要:
We examined the effects of vitamin C supplementation on neutrophil function during exercise loading.Neutrophil functions
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, namely the reactive oxygen species （ROS） production capability, neutrophil phagocytic activity（PA） and serum opsonic activity （SOA） were measured before and after a 2-hour unified loading exercise （ULE）both before and after a 7-day intensified training camp for 22 male judoists. The parameters were assessed neutrophilcount, myogenic enzymes, vitamin C in serum, SOA, PA, ROS production capability, body composition, and so on.Subjects were randomly assigned to two groups; the VC group （daily diet supplementation of 1,500 mg vitamin C）and the Control group （no vitamin C supplementation）. The post-camp pre-ULE vitamin C level was higher in theVC group than in the Control group, though no such difference was seen at pre-camp. As for neutrophil function,although the typical changes seen following a single bout of normal exercise, namely an increase in SOA and ROS,and a decrease in PA, were recorded following the pre- and post-camp ULEs in both groups, significant differencein change rates were not seen between both groups. In conclusion, vitamin C supplementation had no significantinfluence on changes in neutrophil function.
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| 15.
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Maruyama, Masateru ; Odagiri, Hiroki ; Ikenaga, Shojiro-Kazunori ; Nodagashira, Tatsuya ; Sato, Toshiyuki ; Hakamada, Kenichi ; Munakata, Hirofumi
概要:
We examined the therapeutic efficacy for the suicide gene introduction using two recombinant adenovirusvectors with HER2
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promoter and Cre/loxP system in human gastric cancer cell lines, MKN-7 and MKN-28. HER2protein level was more expressed in MKN-7 than in MKN-28. Next, we constructed a Cre recombinae expressionvector in HER2-producing cell specifically, AxHER2NCre and AxCALNCD expressing cytosine deaminase （CD）gene under the control of the CAG promoter by the Cre switching system. Much higher CD messenger RNA （CDmRNA） and CD protein expression were induced in cells by the double infection method than by AxCALNCD only.Furthermore, CD mRNA and CD protein expression were induced higher in HER2-overexpressing cell line, MKN-7 than in MKN-28. We examined the efficacy of cell growth inhibition using 5-fluorocytosine （5-FC） as anti-tumorprodrug. Inhibition effect was dose-dependent at each cell line and rate was more in MKN-7 in comparison withMKN-28. This system can be applied for HER2-overexpressing cancer specific gene therapy.
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| 16.
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Kawaguchi, Yoko ; Hashiba, Eiji ; Kitayama, Masatou ; Hirota, Kazuyoshi
概要:
A 30-year-old woman underwent an emergency cesarean section because of fetal distress involveduterine rupture. As the pl
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acenta was firmly adhered to the uterine wall, following safe delivery, it took over one hour and more than 10000 ml blood loss until the placenta was removed. Additional blood was ordered, but there was delay in delivery. Bleeding was extremely beyond our estimation, and hardly controlled despite of transfusing1400 ml of prepared blood. With stopping operation and continuing hyperoxic ventilation, we decided to start intraoperative blood salvage. Though hemoglobin, platelet, and base excess decreased to 2.7 g/dl, 1000/μl, and -11.2,hemodynamics could be maintained with use of a blood salvage system. Salvaged blood was very helpful to manage critical hemodilution until additional blood was reached. Overall estimated blood loss was 20190 ml and massive blood transfusion was needed. The patient emerged from anesthesia with no neurological complication. Rarely but actually cesarean section resulted in life-threatening hemorrhage. Blood salvage systems is safe and useful during cesareansection. Blood salvage should be usually considered one of the suitable choices in cesarean section patients when supply of homologous blood is limited.
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| 17.
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Oomura, Yutaka ; Aou, Shuji ; Fukunaga, Kouji ; Moriguchi, Sigeki ; Sasaki, Kazuo
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Leptin is well known to be involved in the inhibition of feeding, thermogenesis, reproduction and neuroendocrine functio
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ns through its actions on the rodent hypothalamic receptors. Leptin facilitated the presynaptic transmitter release and postsynaptic sensitivity to the transmitters in the hippocampal CA1 neurons. Thus longterm potentiation （LTP） and the phosphorylation of Ca2+/calmodulin protein kinase II （CaMK II） were facilitated in the CA1 neurons. Therefore behavioral performance related to spatial learning and memory was improved by leptin in vivo applications. Orexin-A produced by glucose-sensitive neurons in the lateral hypothalamic area （LHA）and released during food intake facilitates feeding. Orexin-A suppressed LTP and CaMK II phosphorylation without affecting the presynaptic transmitter release. Therefore behavioral performance on learning and memory was impaired. The present studies suggest that leptin and orexin signalings in the brain may have important implications for cognitive function.
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| 18.
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Herman, Peter ; Sanganahalli, Basavaraju G. ; Coman, Daniel ; Blumenfeld, Hal ; Hyder, Fahmeed
概要:
Neuronal activity mapping of cerebral functions using oxidative energetics has become an accepted functional magnetic re
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sonance imaging （fMRI） technique, termed calibrated fMRI. It requires calculation of oxygen consumption （CMRO2） from blood oxygenation level dependent （BOLD） signal using multi-modal measurements of blood flow （CBF） and volume （CBV）. This approach is based on a biophysical model which describes tissue oxygen extraction at steady-state, therefore it is unclear if this conventional steady-state BOLD model can be applied transiently for calculating dynamic CMRO2 changes. In particular, it is unknown whether calculation of CMRO2 from calibrated fMRI differs between brief and long stimuli. In this study linearity was experimentally demonstrated between BOLD-related components and neural activity. We used multi-modal fMRI （at 11.7T） and neuronal signal measurements of local fi eld potential （LFP） and multi-unit activity （MUA） in α-chloralose anesthetized rats during forepaw stimulation to show that respective transfer functions （of BOLD, CBV, CBF） generated by deconvolution with LFP（ or MUA） are time invariant, for events in the millisecond to minute range. Since the transfer functions are time invariant for event-related and steady-state stimuli, it is possible to use calibrated fMRI in a dynamic manner. The multi-modal results allowed assignment of a significant component of the BOLD signal that can be ascribed to CMRO2 transients. Here we discuss the importance of minimizing residual signal, represented by the diff erence between modeled and raw signals, in convolution analysis using multi-modal fMRI and neural signals.
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| 19.
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Yamada, Katsuya ; Yamamoto, Toshihiro ; Watanabe, Seiji ; Nishiuchi, Yuji ; Teshima, Tadashi ; Matsuoka, Hideaki ; Suga, Sechiko
概要:
Glucose transport activity in mammalian cells has been monitored by radiolabeled tracers such as [14C] 2-deoxy-D-glucose
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. However, due to their limited spatial and temporal resolution, measuring glucose uptake in single, living cells was diffi cult. We have developed a fl uorescent D-glucose derivative, 2-[N（- 7-nitrobenz-2-oxa-1,3-diazol-4-yl）amino]-2-deoxy-D-glucose （2-NBDG）, that allows a more sensitive measurement of glucose uptake in live cells. Indeed, the uptake of 2-NBDG takes place through glucose transporters （GLUTs） in a concentration-, time-, and temperature-dependent manner. Kinetic analysis revealed that apparent Km values for the uptake were similar to those reported by D-glucose and the nonmetabolizable glucose analogue, 3-O-methyl-D-glucose, found in pancreatic islet cells. This method can be combined with Ca2+ imaging and subsequent immunocytochemical identifi cation of cells. So far, 2-NBDG has been used for monitoring glucose uptake into a wide variety of mammalian cells including astrocytes and neurons. However, it has been pointed out that 2-NBDG method requires very accurate procedures, since the fl uorescence intensity is an arbitrary measure. In addition, temporal changes in health of cells in question should be verifi ed throughout the experiment by an independent way such as using patch clamp. To overcome the diffi culty, we synthesized transporter- unrecognizable glucose analogues as control for 2-NBDG uptake. These include 2-[N（- 7-Nitrobenz-2-oxa-1,3-diazol-4-yl）amino]-2-deoxy-L-glucose [2-NBDLG], the antipode of 2-NBDG. A combined use of transporter-recognizable（ D-isomer） and unrecognizable（ L-isomer） fl uorescent analogues allows us to investigate not only net stereospecifi c transport of glucose in live cells but also ligand-transporter interactions and non-transportermediated glucose movement.
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| 20.
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Yamazaki, Daisuke ; Horiuchi, Junjiro ; Saitoe, Minoru
概要:
Age-related memory impairment （AMI） is an important phenotype of brain aging. Understanding the molecular mechanisms und
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erlying AMI is important not only from a scientific viewpoint but also for the development of therapeutics that may eventually lead to developing drugs to combat memory loss. AMI has been generally considered to be an overall or nonspecifi c decay of memory processes that results from dysfunction of neural networks. However, extensive behavioral genetic characterization of AMI with Drosophila demonstrated that AMI results from disruption in specifi c memory process. In Drosophila, memory acquired after a single olfactory conditioning paradigm has three distinct phases: short-term memory （STM）, middle-term memory （MTM）, and longer-lasting anesthesia-resistant memory （ARM）. Significantly, AMI results from the specific decay of only one memory component, amn-dependent MTM, and not other components. Since amnesiac encodes peptides that regulate adenylyl cyclase activity, these studies suggest the importance of the cAMP signaling pathway in AMI in Drosophila, a fi nding consistent with several models of AMI in mammals. In fact, hypomorphic mutations in PKA catalytic subunit signifi cantly suppress AMI. As cAMP signaling is an essential signaling for learning and memory, these studies suggest antagonistic pleiotropic eff ect of cAMP signaling. Due to its short lifespan, powerful genetics, and well-characterized and conserved pathways involved in memory and lifespan, Drosophila will be a useful model system for studying the molecular mechanisms underlying this process.
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| 21.
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Kato, Yukio ; Noshiro, Mitsuhide ; Fujimoto, Katsumi ; Kawamoto, Takeshi
概要:
We cloned Dec1 （Differentiated embryonic chondrocyte-1） and a similar gene, Dec2, in 1997 and 2000, respectively. DEC st
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ructure is similar to that of HES1 and HAIRY, and we observed circadian rhythms of Dec1 mRNA levels in chondrocytes in vitro and rat liver in vivo. We then attempted to fi nd out whether these genes are implicated in the circadian pacemaker: We found that a mutation of Clock abolishes or shifts circadian expression of Dec1 and/or Dec2 in most tissues, including the suprachiasmatic nucleus（ SCN）, and that DEC1 and DEC2 modulate their own circadian expression and that of some other clock genes by auto-regulatory and interlocked feedback mechanisms. Studies on defi ciencies of these genes indicate that Dec1 and Dec2 play roles in the phase shift and maintenance of accurate circadian rhythms and clock outputs to some physiological activities. We speculate that clock genes, including Dec, are involved in the pathology of various diseases and aging.
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| 22.
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Kijima, Hiroshi ; Sato, Fuyuki ; Bhawal, Ujjal Kumar ; Kawamoto, Takeshi ; Fujimoto, Katsumi ; Imaizumi, Tadaatsu ; Imanaka, Tadanobu ; Kondo, Jun ; Koyanagi, Satoru ; Noshiro, Mitsuhide ; Yoshida, Hidemi ; Kato, Yukio
概要:
The circadian rhythms in mammals are regulated by a pacemaker located in the suprachiasmatic nucleus of the hypothalamus
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. Five clock-gene families, i.e. Clock, Bmal, Per, Cry and Dec, have been found to be involved in a transcription-translation feedback loop that generates the circadian rhythm at the intracellular level. In this study, we examined functional analysis of the Dec gene. DEC1 and DEC2 are basic-helix-loop-helix （bHLH） transcription factors, involved in cellular diff erentiation, responses to hypoxia, and circadian rhythms. We recently showed that the expression of DEC1 and DEC2 was upregulated by hypoxia, however, the functions of these two factors under hypoxic conditions have not been elucidated in detail. It is well established that the expression of vascular endothelial growth factor （VEGF） is upregulated by hypoxia, and the expression of VEGF in response to hypoxia depends on transcriptional activation by a heterodimer comprising hypoxia-inducible factor 1 α （HIF-1α） and arylhydrocarbon receptor nuclear translocator 1 （ARNT1）. In the present study, we showed that DEC2, but not DEC1, suppressed VEGF gene expression under hypoxic conditions. DEC2 protein was co-immunoprecipitated with HIF-1α but not with ARNT1. The binding of HIF-1α to the hypoxia response element（ HRE） in the VEGF promoter was decreased by DEC2 overexpression, and increased by DEC2 knockdown. We also showed that the circadian expression of VEGF showed a reciprocal pattern to that of DEC2 in cartilage. DEC2 had a circadian oscillation in implanted Sarcoma 180 cells. We conclude that DEC2 negatively regulates VEGF expression and plays an important role in the pathological conditions in which VEGF is involved.
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| 23.
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Wakamori, Minoru ; Uriu, Yoshitsugu ; Miki, Takafumi ; Kiyonaka, Shigeki ; Mori, Yasuo
概要:
Active zones are highly specialized sites for release of neurotransmitter in presynaptic nerve terminals. The spacing be
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tween voltage-dependent calcium channels（ VDCCs） and synaptic vesicles at active zones is thought to infl uence the dynamic properties of synaptic transmission. Recently we have demonstrated a novel molecular interaction between VDCCs and an active zone scaffolding protein, rab3-interacting molecule 1 （RIM1）. The RIM1 induced a pronounced deceleration of inactivation rate and a depolarizing shift of the inactivation curve of recombinant P/Q-type VDCC expressed as α1Aα2/δβ1a complex in baby hamster kidney cells. During 2-s voltagedisplacement to -30 mV, which is the threshold of the P/Q-type VDCC activation, almost all channels were inactivated in the absence of the RIM1 （closed-state inactivation）, but less than 20% of the channels were inactivated in the presence of the RIM1. Thus, the RIM1 coordinates calcium signaling and spatial organization of molecular constituents at presynaptic active zone. A mutation has been identified for an autosomal dominant cone-rod dystrophy CORD7 in the RIM1 gene. Interestingly, the aff ected individuals showed signifi cantly enhanced cognitive abilities across a range of domains. The mouse RIM1 arginine-to-histidine substitution （R655H）, which corresponds to the human CORD7 mutation, modifi es RIM1 function in regulating VDCC currents elicited by the P/Q-type Cav2.1 and L-type Cav1.4 channels. The data can raise an interesting possibility that CORD7 phenotypes including retinal defi cits and enhanced cognition are at least partly due to altered regulation of presynaptic VDCC currents.
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| 24.
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Itoh, Ken ; Kosaka, Kunio ; Mimura, Junsei ; Satoh, Takumi
概要:
Electrophilic compounds, such as curcumin and neurite outgrowth-promoting prostaglandins （NEPPs）, have been known to enh
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ance neurite outgrowth in the presence of small amounts of nerve growth factor （NGF）in PC12 cells. However, the redox-sensitive molecular target for enhanced neurite outgrowth is largely unknown. NGF exerts its function by binding to the cell surface tyrosine kinase receptor, TrkA. Recently, Shibata et al. reported that PTP1B catalyses the dephosphorylation of TrkA. They also demonstrated that an electrophile from Japanese horseradish Wasabi, 6-methylsulfi nylhexyl isothiocyanate （6-HITC）, inactivates PTP1B causing sustained phosphorylation and activation of TrkA and promoting neural diff erentiation of PC12 cells. Further, we recently discovered that carnosic acid （CA）, a catechol-type electrophilic compound that is a major ingredient in the herb rosemary, strongly promotes neurite outgrowth of PC12h cells by activating the redox-sensitive transcription factor, Nrf2. Activation of Nrf2 by CA caused a marked induction of p62/ZIP, an important signaling scaff old protein for NGF signaling. Unexpectedly, NGF also activates Nrf2, which is essential for NGF-mediated neural diff erentiation. Thus, electrophiles may enhance neural diff erentiation by both TrkA-dependent and -independent mechanisms.
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| 25.
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Nakabeppu, Yusaku ; Sheng, Zijing ; Oka, Sugako
概要:
Oxidative DNA lesions, such as 8-oxoguanine（ 8-oxoG）, accumulate in nuclear and mitochondrial genomes during aging, and
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such accumulation is known to dramatically increase in patient brains with Parkinson’s disease（ PD）or Alzheimer’s disease（ AD）. To counteract oxidative damage to nucleic acids, human and rodents are equipped with three distinct enzymes, MTH1, OGG1 and MUTYH. MTH1 hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP to their monophosphate forms. OGG1 and MUTYH are DNA glycosylases excising 8-oxoG opposite cytosine and adenine opposite 8-oxoG in DNA, respectively. We showed a signifi cant increase in 8-oxoG in cellular DNA as well as altered expression of MTH1, OGG1 and MUTYH in PD and AD brains, suggesting that the buildup of 8-oxoG may cause neurodegeneration. We have shown that buildup of 8-oxoG in either nuclear or mitochondrial DNA causes MUTYH-dependent cell death through two distinct pathways, and that accumulation of oxidized nucleotides in nucleotide pools also causes MUTYH-dependent cell death. MTH1-null mice exhibited an increased buildup of 8-oxoG in striatal mitochondrial DNA followed by more extreme neuronal dysfunction after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration, while hMTH1-transgenic mice are resistant to a mitochondrial neurotoxin, 3-nitropropionic acid （3-NP）-induced striatal degeneration, in comparison to wild-type mice. We found that doubleknockout （DKO） mice lacking OGG1 and MTH1, and to a lesser extent OGG1-KO mice, are signifi cantly sensitive to 3-NP-induced striatal degeneration, in comparison to MTH1-KO or wild-type mice, while MUTYH defi ciency increases resistance to 3-NP in OGG1-KO or wild-type background. We thus demonstrated that 8-oxoG accumulated in brain genomes causes neurodegeneration in a MUTYH-dependent manner, and which is effi ciently suppressed by MTH1 and OGG1.
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| 26.
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Wakabayashi, Koichi ; Miki, Yasuo ; Tanji, Kunikazu ; Mori, Fumiaki
概要:
α-Synucleinopathies comprise a group of neurodegenerative disorders that share α-synuclein （αS）accumulation in selected
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vulnerable neurons and glia, i.e. Parkinson’s disease （PD）, dementia with Lewy bodies and multiple system atrophy（ MSA）. The histological hallmark of PD is neuronal αS aggregates called Lewy bodies（ LBs）. LB formation has been considered to be a marker for neuronal degeneration, because neuronal loss is found in the predilection sites for LBs. However, recent studies have suggested that oligomers and protofi brils of αS are cytotoxic, and that LBs may represent a cytoprotective mechanism in PD. The histological hallmark of MSA is αS aggregates in the oligodendrocytes referred to as glial cytoplasmic inclusions（ GCIs）. αS inclusions are also found in the neuronal somata, axons and nucleus. At present, two degenerative processes have been considered in this disease; one is due to the widespread occurrence of GCIs associated with oligodendroglia-myelin degeneration, and the other is due to the aggregation of αS in neurons in several brain regions. These two processes might synergistically cause neuronal depletion in MSA.
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| 27.
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Tanji, Kunikazu ; Mori, Fumiaki ; Wakabayashi, Koichi
概要:
NEDD8 ultimate buster-1 （NUB1） is a potent down-regulator of the ubiquitin-like protein NEDD8, because it directly inter
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acts with NEDD8 and targets it and its conjugates to the 26S proteasome for proteolytic degradation. Recently, we found that NUB1 physically interacts with synphilin-1 through its NEDD8-binding site, implying that NUB1 also targets synphilin-1 to the 26S proteasome for proteolytic degradation. Synphilin-1 is an α-synuclein-interacting protein and is a major component of inclusion bodies found in the brains of patients with neurodegenerative α-synucleinopathies, including Parkinson’s disease. In our recent studies, we immunostained sections of brains from patients with Parkinson’s disease and other α-synucleinopathies and demonstrated that NUB1, as well as synphilin-1, accumulates in the inclusion bodies. To defi ne the role of NUB1 in the formation of these inclusion bodies, we performed a co-transfection assay using cultured HEK293 cells. This assay showed that NUB1 suppresses the formation of synphilin-1-positive inclusions. Further biochemical assays revealed that NUB1 overexpression leads to the proteasomal degradation of synphilin-1. These results and our previous observations suggest that NUB1 indeed targets synphilin-1 to the proteasome for its effi cient degradation, which, because of the resultant reduction in synphilin-1, suppresses the formation of synphilin-1-positive inclusions. In addition to these basic science aspects, our fi ndings on NUB1 have two important bearings clinically. First, they suggest that NUB1 could serve as a neuropathological marker in patients with α-synucleinopathies because it is strongly accumulated with synphilin-1 in the inclusions of their brain cells. Second, they suggest that NUB1 could be a potential therapeutic target for α-synucleinopathies.
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| 28.
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Younkin, Steven
概要:
Like my presentation, this article focuses exclusively on the role of the amyloid ß protein in Alzheimer’s Disease. I fi
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rst provide an overview of the research fi ndings that have made Aß, more specifi cally Aß42, a compelling therapeutic target for AD that is being pursued by virtually all pharmaceutical companies. I then discuss specifi capproaches to Aß-centered therapy. It is widely believed that the best way to manage AD will be through preventive therapy. To implement preventive therapy, it is critically important to be able to identify normal subjects who are at imminent risk for AD. In the last part of this article, I review recent evidence indicating that the measurement of Aß40 and Aß42 in plasma may be useful for identifying those normal subjects who will develop AD.
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| 29.
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Mori, Hiroshi ; Tomiyama, Takami ; Ishibashi, Kennichi ; Ohnishi, Kiyouhisa ; Teraoka, Rie ; Fukushima, Akiko ; Takuma, Hiroshi ; Shimada, Hiroyuki ; Ataka, Suzuka ; Umeda, Tomohiro ; Kitajima, Erika ; Fujita, Yuki ; Yamashita, Yuki ; Yamamoto, Keiichi ; Miki, Takami ; Matsuyama, Shogo ; Iso, Hiroyuki ; Nagata, Tetsu ; Nishizaki, Tomoyuki ; Wada, Yasuhiro ; Yoshioka, Eito ; Watanabe, Yasuyoshi
概要:
Alzheimer's disease (AD) is the major and common disease usually for aged people to show progressive neurodegenerative d
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isorder with the dementia. Amyloid-beta （also β-protein and referred here to as Aβ） is a wellestablished seminal peptide in AD that is produced from the amyloid precursor protein （APP） by consecutive digestions with β-secretase of BACE and gamma-secretase of the presenilin complex. Abnormal cerebral accumulation of Aβ such as insoluble fi brils in senile plaques and cerebral amyloid angiopathy （CAA） are observed as a neuropathological hallmark of AD. In contrast to such insoluble fi brillary Aβ, a soluble oligomeric complex is discussed as ADDLs, Aβ oligomer, low-n oligomer Aβ, Aβ*56 or so. Despite their diff erent names, it is proposed as the current hypothesis that oligomeric Aβ is the direct molecule to cause synaptic toxicity and cognitive dysfunction in the early stages of AD. We identifi ed a novel APP mutation （E693delta; referred to as the Osaka mutation） in a pedigree with probable AD resulting in a variant Aβ lacking glutamate at position 22. Based on theoretical prediction and in vitro studies on synthetic mutant Aβ peptides, the mutated Aβ peptide showed a unique aggregation property of enhanced oligomerization but no fi brillization. This was further confi rmed by PiB-PET analysis on the proband patient. Collectively together, we conclude that the Osaka mutation is the fi rst human evidence for the hypothesis that oligomeric Aβ is involved in AD.
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| 30.
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論文
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Ghiso, Jorge ; Tomidokoro, Yasushi ; Revez, Tamas ; Frangione, Blas ; Rostagno, Agueda
概要:
Cerebral amyloid angiopathy （CAA） is increasingly recognized as a major contributor of Alzheimer’s disease（ AD） pathogen
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esis. To date, vascular deposits and not parenchymal plaques appear more sensitive predictors of dementia. Amyloid deposition in and around cerebral blood vessels plays a central role in a series of response mechanisms that lead to changes in the integrity of the blood-brain barrier, extravasations of plasma proteins, edema formation, release of inflammatory mediators and matrix metalloproteases which, in turn, produce partial degradation of the basal lamina with the potential to develop hemorrhagic complications. The progressive buildup of amyloid deposits in and around blood vessels chronically limits blood supply and causes focal deprivation of oxygen, triggering a secondary cascade of metabolic events several of which involve the generation of nitrogen and oxygen free radicals with consequent oxidative stress and cell toxicity. Many aspects of CAA in early- and lateonset AD ‒the special preference of Aβ40 to deposit in the vessel walls, the favored vascular compromise associated with many Aβ genetic variants, the puzzling observation that some of these vasculotropic variants solely manifest with recurrent hemorrhagic episodes while others are mainly associated with dementia‒ await clarifi cation. Non-Aβ cerebral amyloidoses reinforce the viewpoint that plaque burden is not indicative of dementia while highlighting the relevance of non-fi brillar lesions and vascular involvement in the disease pathogenesis. The lessons learned from the comparative study of Aβ and non-Aβ cerebral amyloidosis provide new avenues and alternative models to study the role of amyloid in the molecular basis of neurodegeneration.
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| 31.
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Carrasquillo, Minerva M. ; Zou, Fanggeng ; Pankratz, V. Shane ; Wilcox, Samantha L. ; Ma, Li ; Walker, Louise P. ; Younkin, Samuel G. ; Younkin, Curtis S. ; Younkin, Linda H. ; Bisceglio, Gina D. ; Ertekin-Taner, Nilufer ; Crook, Julia E. ; Dickson, Dennis W. ; Peterson, Rnald C. ; Graff-Radford, Neill R. ; Younkin, Steven G.
概要:
By analyzing late onset Alzheimer’s disease （LOAD） in a genome wide association study of 3 American Caucasian series and
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evaluating the 25 SNPs with most significant allelic association in 4 additional series, we identifi ed a SNP （rs5984894） on Xq21.3 in PCDH11X that is strongly associated with LOAD in American Caucasians （total n=4,855; AD:2,391; control:2,464）. Analysis of rs5984894 by logistic regression using sex as a covariate gave a global p value of 3.9 x 10-12 in the combined series. Odds ratios were 1.75（ 95% CI 1.42-2.16） for female homozygotes （P=2.0x10-7） and 1.26 （95% CI 1.05-1.51） for female heterozygotes （P=0.01） compared to female non-carriers. For male hemizygotes （P=0.07） compared to male non-carriers the odds ratio was 1.18 （95% CI 0.99-1.41）. The eff ect of this variant was dose dependent, as females homozygotes for the minor allele were at signifi cantly greater risk than heterozygous females and hemizygous males. We tested additional variants in PCDH11X for association with LOAD. One of these variants, rs2573905, showed association with LOAD similar to that for rs5984894, albeit with a slightly more significant p-value （5.4x10-13）. This is not surprising since these two variants are in near perfect linkage disequilibrium （r2 = 0.98, D’ = 0.99）, and the minor allele of these two SNPs occur on the same haplotype. However, rs2573905 is in a sequence that has been evolutionarily conserved between human and mice, with 70% sequence identity over 100bp, suggesting a possible functional role for this SNP. Joint analysis of APOE and rs2573905 genotypes showed that 75 women with LOAD （4.9%） were homozygous both for APOE e4 and for rs2573905 whereas only 2 unaff ected women （0.15%）, both age 73, were double homozygotes. These fi nding suggest that the double homozygote may be fully penetrant in women over the age of 73 and that this combination may account for ~5% of the AD that occurs in women.
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| 32.
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Shoji, Mikio
概要:
Antibodies to amyloid β protein（ Aβ） are present naturally or after Aß vaccine therapy in human plasma. To clarify their
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clinical role, we examined plasma samples from 113 patients with Alzheimer’s disease（ AD） and 205 normal controls using the tissue amyloid plaque immunoreactivity （TAPIR） assay. A high positive rate of TAPIR was revealed in AD （45%） and age-matched controls （41%）, however, no signifi cance was observed. No signifi cant difference was observed in the MMS score or disease duration between TAPIR-positive and negative samples. TAPIR-positive plasma reacted with the Aß40 monomer and dimer, and the Aβ42 monomer weakly, but not with the Aβ42 dimer. TAPIR was even detected in samples from young normal subjects and young Tg2576 transgenic mice. Although the Aβ40 level and Aβ40/42 ratio increased, and Aβ42 was significantly decreased in plasma from AD groups when compared to controls, no signifi cant correlations were revealed between plasma Aß levels and TAPIR grading. Thus an immune response to Aβ40 and immune tolerance to Aβ42 occurred naturally in humans without a close relationship to the Aβ burden in the brain. Clarifi cation of the mechanism of the immune response to Aβ42 is necessary for realization of an immunotherapy for AD.
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| 33.
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Shimamura, Norihito ; Ohkuma, Hiroki
概要:
We developed a novel trans-femoral artery approach to the rat middle cerebral artery occlusion model （TF-MCAO） without s
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acrifi cing the external carotid artery （ECA） with/without the pterygopalatine artery, which is important for chewing food. To make the TF-MCAO we fi rst dissect the left common carotid artery（ CCA）, ECA, and internal carotid artery（ ICA）. Transient occlusion clips are applied to the proximal ECA and the pterygopalatine artery; we never sacrifi ce the ECA branch. A 24-gage catheter is inserted into the left femoral artery. We insert a slightly bent 0.014 inch hydrophilically-coated guide wire via a haemostasis valve. Anatomically, the left common carotid artery is located rostral to the descending aorta, permitting a straight-forward, blind approach to the CCA. The guide wire is gently advanced about 17 mm from the bifurcation of the CCA until slight resistance is encountered. The guide wire and temporary occlusion clips are withdrawn after 90 minutes. Rats were sacrifi ced 24h after reperfusion. Eleven rats were examined. One rat died before occlusion due to deep anesthesia. The success rate for producing infarction was 80%. The mean infarction volume of the basal ganglia was 94.4 ㎣ ± 9.4 se and mean infarction volume of the cerebral cortex was 124.2 ㎣ ± 21.6 se. No rat died due to cerebral infarction and no rat suff ered subarachnoid hemorrhage. We conclude that TF-MCAO was useful for producing a cerebral infarction.
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| 34.
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Yoshida, Hidemi ; Mimura, Junsei ; Imaizumi, Tadaatsu ; Matsumiya, Tomoh ; Ishikawa, Akira ; Metoki, Norifumi ; Tanji, Kunikazu ; Ota, Ken ; Kosaka, Kunio ; Itoh, Ken ; Satoh, Kei
概要:
Optimization of neuronal function and survival is an important goal in the treatment of cerebrovascular diseases in orde
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r to avoid or improve devastating long-term sequelae. Nerve growth factor （NGF） is essential for neuronal growth and survival in the central nervous system （CNS）. Vascular endothelial growth factor （VEGF） is a potent mitogen specifi c for endothelial cells and a stimulator of neovascularization. VEGF also enhances vascular permeability, which may promote the development of brain edema during cerebral ischemia. These molecules aff ect the outcome of ischemia/reperfusion injury in the CNS. Edaravone, a brain-penetrant, free radical scavenger, is known to ameliorate postischemic neuronal dysfunction. Transcription factor Nrf2（ nuclear factor-erythroid 2-related factor 2）, a master regulator of antioxidant responses, plays an important role in the coordinated expressions of stress-inducible genes. Astrocytes express various genes involved in the regulation of neuronal functions, and the regulation of astrocyte gene expressions may be a potential therapeutic target in brain injury. This review aims to appraise the eff ects of radical scavenger edaravone and a natural Nrf2-inducer as neuroprotective agents in human astrocytes, particularly under an experimental model for hypoxia/reoxygenation.
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| 35.
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Cahill, Julian ; Zhang, John H.
概要:
Background ̶Subarachnoid hemorrhage （SAH） remains a disease without any definitive treatment options. Research, to date,
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has concentrated on the pathophysiology of vasospasm. Recent evidence supports the concept of Early Brain Injury （EBI）, a phenomena which may help to explain the complex pathophysiology seen in patients after a SAH. Summary ̶EBI aims to describe the pathophysiological events that occur in the brain within the first seventy two hours after a SAH, before the onset of vasospasm. A number of pathways have been identifi ed which may play a role in the etiology of EBI. This review provides a brief synopsis of EBI and its implications for the future. Conclusions ̶EBI may represent a key event in the development of both vasospasm and Delayed Ischemic Neurological Deficit （DIND） after subarachnoid hemorrhage. Additional studies are required to determine the pathophysiology of EBI and to examine its role as a possible precursor to both vasospasm and DIND.
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| 36.
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論文
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Urade, Yoshihiro
概要:
Prostaglandin（ PG） D2 is the most potent endogenous sleep-promoting substance thus far reported and its sleep-inducing m
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echanism is the best characterized at the molecular level. PGD2 is produced by lipocalin-type PGD synthase （L-PGDS） localized in the leptomeninges, choroid plexus, and oligodendrocytes in the brain and is secreted into the cerebrospinal fluid as a sleep hormone. PGD2 stimulates DP1 receptors localized in the arachnoid membrane at the basal forebrain to release adenosine as a paracrine sleep-promoting molecule, which activates adenosine A2A receptor-expressing neurons located in the basal forebrain. These cells subsequently excite the sleep-active neurons in the ventrolateral preoptic area and concomitantly suppress the tuberomammillary nucleus, a histaminergic arousal center, through GABAergic and galaninergic inhibitory projection, to induce sleep. The administration of a PGD synthase inhibitor （SeCl4）, DP1 antagonist （ONO-4127Na） or adenosine A2 receptor antagonist （caffeine） induces insomnia, indicating that the PGD2-adenosine system is crucial for the maintenance of physiological sleep.
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| 37.
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論文
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Yamazaki, Yoshihiko ; Hozumi, Yasukazu ; Kaneko, Kenya ; Fujiwara, Hiroki ; Kato, Hiroshi ; Fujii, Satoshi
概要:
Action potentials are the fundamental signals for relaying information from one region to another in the nervous system.
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Action potentials are propagated along axons without decrease of their amplitudes and are conducted with constant velocity depending on axonal diameter and myelin. It is considered that the modulation of firing patterns of action potentials in the neural circuit infl uences the information processing in the brain. We investigated the modulatory eff ects of glial cells on the fi ring pattern and the axonal conduction of action potentials using rat hippocampal slice preparation. In our previous study, we focused on interneuron / perineuronal glial cell pairs in CA1 region and reported that perineuronal glial cells could be classifi ed into two groups, one group belong astrocytes （perineuronal astrocytes） and the other group oligodendrocytes （perineuronal oligodendrocytes）, based on their membrane properties and immunohistochemical study. Direct depolarization of perineuronal astrocytes modulated the directly induced fi ring pattern of the interneuron, with initial facilitation and subsequent suppression. We also studied the oligodendrocytes in the alveus and examined their modulatory effects on the conduction of action potentials along axons of CA1 pyramidal cells. Direct repetitive depolarization of oligodendrocytes shortened the latencies of action potentials evoked by antidromic stimulation. These results indicate that glial cells infl uence the firing pattern and axonal conduction of action potentials, and that their effects involve both facilitation and suppression.
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| 38.
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論文
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Masamoto, Kazuto ; Obata, Takayuki ; Kanno, Iwao
概要:
Cerebral blood flow is tightly regulated, and local metabolic demands are met by adjustments to the regional density of
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microvascular networks and by temporal and spatial changes in microvascular blood flow. Cerebral blood flow regulation may involve communication between and across the vascular cells and neural or glial cells in either rapid or slow conduits. In the present study, we report distinct diff erences between the dynamic reactions of cerebral arterial networks in cortical surface and in intracortical tissue regions in response to sensory stimulation. Using confocal and multi-photon excitation laser scanning fluorescence microscopy, we imaged the cortical surface and subsurface vascular networks in the somatosensory cortex of isoflurane-anesthetized rats. Changes in lumen diameter were imaged at a rate of 13 frames per second with a fi eld of view of 512 by 512 pixels. We consistently observed a stimulus-dependent increase in the lumen diameter of arterial networks in both cortical surface and subsurface regions. The onset time of vasodilation was observed to be ~0.8 sec for the subsurface arterioles（ <40 μm）, which was signifi cantly shorter than the ~1.1 sec vasodilation onset time of the surface arteries （20-120 μm）. The peak dilation accounted for 10% of the pre-stimulus baseline diameter. Further, the propagation of surface arterial vasodilation increased in a stimulus-dependent manner. The results indicate that global vasodilation of upstream parent arteries may be necessary to prevent “blood steal” by inactive regions nearby. Further studies are needed to elucidate the physiological mechanisms underlying the propagation of vasodilation induced by neural stimulation.
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| 39.
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論文
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Yamamoto, Toshihiro ; Nishiuchi, Yuji ; Teshima, Tadashi ; Watanabe, Seiji ; Suga, Sechiko ; Matsuoka, Hideaki ; Yamada, Katsuya
概要:
D-Glucose is one of the most important energy sources for the survival of various organisms, from E. coli to mammals. Fo
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r live-cell monitoring of glucose uptake at the single-cell level, a fl uorescent D-glucose derivative 2-[N-（7-nitrobenz-2-oxa-1,3-diazol-4- yl）amino]-2-deoxy-D-glucose [2-NBDG], which we developed, has been widely used in various research fi elds. For the last ten years, however, researchers have awaited an optical control substance for evaluating the extent of non-specifi c adsorption of 2-NBDG upon plasma membrane and/or the rate of unhealthy 2-NBDG uptake through partially（ or transiently） damaged membrane. Here we introduce a fluorescent L-glucose derivative, 2-[N（- 7-Nitrobenz-2-oxa- 1,3-diazol-4-yl）amino]- 2-deoxy-Lglucose [2-NBDLG]. L-Glucosamine is a key intermediate toward the synthesis of 2-NBDLG, but not commercially available. Although a few papers on the synthesis of L-glucosamine have been reported, a new synthetic method of L-glucosamine should be absolutely required in practical view of optical purity and preparative scale. We converted commercially available L-mannose into desired L-glucosamine by 10 steps in 14% of overall yield. The 1H-NMR data of synthetic L-glucosamine were completely identical with those of commercially available D-glucosamine. On the other hand, optical purity of L-glucosamine was confi rmed by comparison of specifi c rotation with that of D-glucosamine. L-Glucosamine thus obtained was coupled with NBD-halide to give 2-NBDLG. Use of transporterrecognizable （D-isomer） and unrecognizable （L-isomer） fluorescent analogues combined with real-time confocal microscopy, should provide valuable information on dynamism of glucose transport.
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| 40.
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論文
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Takuwa, Hiroyuki ; Masamoto, Kazuto ; Obata, Takayuki ; Kanno, Iwao
概要:
The mechanism regulating cerebral blood fl ow （CBF） during brain function （neurovascular coupling）has been widely invest
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igated in animals under anesthetized conditions, though anesthesia is known to greatly aff ect neurovascular physiology. The present study aims to develop a novel model for neurovascular coupling studies in awake-behaving mice. Male C57BL/6J mice were initially anesthetized with isofl urane in preparation for attaching the study apparatus to the head. The animal was tethered to the study apparatus but allowed to move spontaneously on a floated ball. The animal behavior and regional CBF in the somatosensory barrel cortex were simultaneously measured with optical motion sensor and laser-Doppler fl owmetry （LDF）, respectively. Anesthesia was discontinued during recovery, while whisker stimulation （frequency 10 Hz and duration 10 or 20 sec） was induced at either the contralateral or ipsilateral side of the LDF recording site. During the experiments, the animals showed no signs of struggling against the head restraint. The intensity of baseline CBF was higher while the animal was under 2% isoflurane aesthesia than it was after anesthesia was stopped. CBF response to stimulation was not observed under anesthesia. After the animal was recovered from anesthesia, an increase in CBF （34 ± 18%） was observed during contralateral stimulation but not during ipsilateral stimulation. The fl uctuation levels of CBF baseline during resting and walking conditions were ±2.7% and ±3.5%, respectively. We observed that these fl uctuations were not due to vibration noises caused by such as air-puff and animal motion in our experimental conditions.
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| 41.
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Munakata, Akira
概要:
OBJECTIVE: It is hypothesized that free radical reactions evoked by oxyhemoglobin （oxyHb） cause cerebral vasospasm after
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aneurysmal subarachnoid hemorrhage （SAH）, even though the detailed mechanisms have not yet been fully established. The aims of this study were thus to investigate, through the use of the doublehemorrhage rabbit model, the possibility that using a potent free radical scavenger, edaravone, will show amelioration of cerebral vasospasm, and to delineate the mechanism of signal transduction that causes cerebral vasospasm.METHODS: In the SAH group, SAH was simulated using the double-hemorrhage rabbit model. In the treatment group, edaravone （0.6 mg/kg） was injected into the central ear vein. Ninety-six hours after SAH, the basilar artery was excised. The degree of cerebral vasospasm was evaluated by measuring the diameter of each basilar artery, and the expression of Rho-kinase in the vascular wall was examined by Western blotting. RESULTS: The diameter of the basilar artery in the edaravone-treated group was 0.71 ± 0.06 mm, which was statistically signifi cantly larger than that in the nontreated SAH group （0.50 ± 0.03 mm; P < 0.01）. The expression of Rho-kinase in the edaravonetreated group was statistically signifi cantly reduced in comparison to that of the nontreated SAH group （P < 0.01）.CONCLUSION: Results from this study indicate that edaravone may potentially serve as an agent in the prevention of cerebral vasospasm in patients after SAH. In addition, it might be suggested that the free radical reaction mediated by oxyHb is concerned with the regulation of the Rho/Rho-kinase pathway.
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| 42.
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Zhang, Hai-xin ; Tanji, Kunikazu ; Yoshida, Hidemi ; Hayakari, Makoto ; Mori, Fumiaki ; Wakabayashi, Koichi
概要:
TDP-43 proteinopathy （amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclus
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ions） is a newly categorized group of neurodegenerative disorders characterized by abnormal accumulation and mislocalization of nuclear TDP-43 protein in the neuronal cytoplasm. 15-deoxy-Δ12,14-prostaglandin J2 （15d-PGJ2） is non-enzymatically produced from PGD2, and plays roles in infl ammation and oxidative stress responses. Indeed, 15d-PGJ2 is up-regulated in the spinal motor neurons in ALS. In this study, biochemical and fluorescent staining analyses showed that 15d-PGJ2 modifi es expression, solubility, and subcellular localization of TDP-43. This alteration was at least partly related to a cyclopentenone ring structure containing an electrophilic carbon of 15d-PGJ2, because 15d-PGJ2 analogue, which lacks an cyclopentenone ring structure, had almost no eff ect on TDP-43 protein. Finally in vitro binding experiment indicated that 15d-PGJ2 is covalently bound to TDP-43 protein. These fi ndings suggest that a sustained high level of 15d-PGJ2 is involved in the pathogenesis of neurodegenerative disorders related to abnormal TDP-43 protein.
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| 43.
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Mori, Fumiaki ; Tanji, Kunikazu ; Miki, Yasuo ; Wakabayashi, Koichi
概要:
Cystatin C （CC）, a cysteine protease inhibitor involved in protein degradation, is a marker of Bunina bodies in lower mo
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tor neurons in amyotrophic lateral sclerosis （ALS）. TDP-43-immunoreactive inclusions are also histological hallmark of ALS. However, immunohistochemical localization of CC in ALS motor neurons with or without inclusions is uncertain. Recently, we demonstrated that the majority of anterior horn cells showed moderate to intense immunoreactivity for CC in controls and that CC immunoreactivity was severely decreased in anterior horn cells in ALS. The proportion of CC-immunolabeled anterior horn cells was reduced regardless of whether those neurons contained Bunina bodies or not. In contrast, the proportion of CC-immunolabeled anterior horn cells was signifi cantly reduced owing to the presence of TDP-43 inclusions. These fi ndings suggest that the formation of TDP-43 inclusions, but not of Bunina bodies, reduces the content of CC in spinal motor neurons and that perturbations in endogenous levels of CC in neurons may participate in neurodegenerative process in ALS.
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| 44.
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Hu, Dong-Liang ; Omoe, Katsuhiko ; Sashinami, Hiroshi ; Shinagawa, Kunihiro ; Nakane, Akio
概要:
Background. Staphylococcal enterotoxins （SEs） are the most common cause of food-borne diseases and toxic shock syndrome
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throughout the world. However, little is known about the mechanism of emesis induced by SEs and no vaccine that prevents SE-induced emesis has been described. Methods. A nontoxic mutant SEA, SEAD227A, was constructed by site-directed mutagenesis and purified from Escherichia coli expression system. House musk shrews, a small emetic animal model, were immunized with SEAD227A and then challenged with wild-type SEA. SEA-induced emesis was recorded for 3 h. Antibody production was analyzed by gel double-immunodiff usion assay. Neutralizing activities of the antibodies to superantigenic and emetic activities were analyzed in vitro and in vivo. Results. SEAD227A was devoid of both superantigenic and emetic activities, but still retained its immunological activity. Immunization with SEAD227A strongly induced specifi c antibody production and signifi cantly provided the protection against SEA-induced emesis. The antibodies from immunized shrews markedly inhibited the SEA-induced proliferation of spleen cells and also significantly ablated the SEA-induced vomiting in the animals. Conclusions. These results suggest that vaccination with SEAD227A devoid of toxic properties provides protection against SEAinduced emesis. This nontoxic mutant and its specifi c antibodies might be useful in the prevention and treatment of staphylococcal food poisoning
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| 45.
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Sashinami, Hiroshi ; Hu, Dong-Liang ; Li, Sheng-Jun ; Mitsui, Toshihito ; Hakamada, Kenichi ; Ishiguro, Yoh ; Fukuda, Shinsaku ; Nakane, Akio
概要:
We investigated the eff ect of p60 that is one of virulence factors of Listeria monocytogenes on host immune response in
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vitro and in vivo. C57BL/6 mice immunized with p60 showed antigen-specifi c T-helper I type immune response. Mouse macrophage RAW264.7 cells produced tumor necrosis factor alpha （TNF-α）, interleukin-12 and interferon beta （IFN-β） in response to stimulation with recombinant p60. Administration of p60 prior to a sublethal infection with L. monocytogenes enhanced innate host resistance in naïve mice. TNF-α and IFN-β production from RAW264.7 cells and bone marrow-derived macrophages were Toll-like receptor 4 （TLR4）-dependent. The enhanced clearance of L. monocytogenes by p60 administration was not shown in C3H/HeJ mice. Our fi ndings demonstrated that p60 enhances host resistance against L. monocytngenes infection through both activation and attenuation of host innate immune response in the TLR4-dependent manner.
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| 46.
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Osanai, Arihiro ; Li, Sheng-Jun ; Nakane, Akio
概要:
Peptidoglycan recognition proteins （PGRPs） are pattern recognition receptors which are conserved from insects to humans.
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PGRPs can recognize bacteria and their cell wall components, peptidoglycans. Human PGRPs have bactericidal activities that can be accomplished by their amidase activities. Insect PGRPs consist of 16 subtypes and some have eff ector functions as the activation of Toll pathway （PGRP-SA）, the activation of Imd pathway （PGRPLC）and induction of autophagosome formation （PGRP-LE）, thus contribute to the elimination of bacteria. However, the role of mammalian PGRPs in bacterial infection remains unclear. In this study, we report the function of mouse PGRP-S, the homologue of PGRP-SA, in bacterial infection. The recombinant protein was produced in Escherichia coli overexpression system and used for specifi cantibody production. We investigated the role of PGRP-S in infection with Listeria monocytogenes, a facultative Gram-positive bacterium that can grow intracellularly. The administration of recombinant PGRP-S before L. monocytogenes infection decreased the number of bacteria in the organs of infected mice. When endogenous PGRP-S was neutralized by antibodies specifi c to PGRP-S, the bacterial number increased. The levels of proinfl ammatory cytokines that are essential in the protection against L. monocytogenes infection were lower when the specific antibody was administered prior to infection. Together with these, it is suggested that PGRP-S plays a role in the protection against L. monocytogenes infection.
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| 47.
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Narita, Koji ; Hu, Dong-Liang ; Nakane, Akio
概要:
Clumping factor A （ClfA） is an adhesin of Staphylococcus aureus, and the residues 40-559 of ClfA （ClfA40-559） compose f
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i brinogen binding domain. It was reported that antibody to ClfA40-559 plays an important role in the protective eff ects. In this study, we investigated the role of IL-17 in the protective eff ects of an immunization with ClfA40-559 against S. aureus infection. Mice immunized or non-immunized with ClfA40-559 were challenged with S. aureus. Bacterial numbers in the organs and survival rates were evaluated. RAW264.7, DC2.4 and spleen cells of naïve and immunized mice were stimulated with ClfA40-559. Cytokine production and mRNA expression in the spleens and cells was determined by ELISA and/or RT-PCR. The survival rate was improved and the bacterial numbers in the organs were reduced in the mice immunized with ClfA40-559. ClfA40-559 induced IL-23p19 mRNA expression and IL-6 but not IL-12 production in DC2.4 and RAW264.7 cells. RORγt and IL-17A mRNA expression in the spleen cells of naïve mice was induced. In the spleen cells of immunized mice, ClfA40-559 induced the high production of IL-17 but not IFN-γ or IL-4. IL-17A, IL-6 and CXCL2 mRNA expression was also increased in the organs of immunized and challenged mice. These results suggest that an immunization with ClfA40-559 induces Th17, and that IL-17 contributes to host defense in early phase of S. aureus infection by promoting neutrophil recruitment.
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| 48.
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Meguro, Reiko ; Asano, Yoshiya ; Odagiri, Saori ; Li, Chengtai ; Shoumura, Kazuhiko ; Ichinohe, Noritaka
概要:
Iron is essential for a variety of cellular functions. However iron, especially ferrous form, has the potential for cata
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lyzing the Fenton reaction to generate highly cytotoxic hydroxyl radicals. Therefore the amount of iron in the brain is strictly controlled. In this study we focused on the cellular and subcellular localizations of nonheme ferric （Fe（III）） and ferrous （Fe（II）） iron in the adult female rat brain by light and electron microscopic histochemistry. Although Fe（II） deposition was much less dominant than Fe（III）, the brain contained iron in both forms. Among the cellular elements of the brain, oligodendrocytes were numerically most prominent and heavily iron-storing cells. Pericapillary astrocytes and sporadic microglial cells also showed dense iron accumulation. Large neurons involved in the motor system were relatively strongly iron-positive. Subcellularly, Fe（III） and Fe（II） were mainly localized in lysosomes, and occasionally in the cytosol and mitochondria. Fe（II）-specific deposit was shown on the luminal membrane of endothelium. With advancing age, both Fe（III） and Fe（II） became distributed more extensively, and accumulated more numerously into oligodendrocytes.
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| 49.
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Maruyama, Ayumi ; Kanazawa, Sachiko ; Shimoyama, Ritsuko ; Hosoi, Kazuhiro ; Hayakari, Makoto
概要:
Although animal studies suggest that centrally active angiotensin-converting enzyme （ACE） inhibitors may protect agains
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t dementia beyond HTN control, the mechanism（s） underlying these improvements in cognitive function remains unclear. We measured the brain peptide levels in rat treated with captopril（ 50 mg/kg） for 3 weeks by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry （SELDI TOF-MS）. Two protein chip arrays were used for peptide profi ling： one with a strong anion- exchanger and the other with a weak cation-exchanger. Comparing with control group, 15 mass peaks were considered specifi c to experimental animals, and 6 peaks were signifi cantly up-regulated and 5 down-regulated.
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| 50.
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Migita, Keisuke ; Honda, Kenji ; Yamada, Junko ; Takano, Yukio ; Ueno, Shinya
概要:
Painful diabetic neuropathy causes allodynia and does not respond to commonly used analgesics such as non-steroidal anti
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-infl ammatory drugs or opioids at doses below those producing disruptive side eff ects. In the present study, we examined the effect of P2X receptor antagonists, which are known to modulate the pain pathway, on mechanical allodynia in streptozotocin（ STZ）-induced diabetic mice. The paw withdrawal frequency measured by von Frey fi laments, began to signifi cantly increase 5 days after STZ injection and was maintained for more than 14 days. Intrathecal administration of P2X receptor antagonists （PPADS and TNP-ATP） inhibited the mechanical allodynia in diabetic mice. Next, the levels of mRNA for the P2X receptors in DRG in diabetic mice were measured using quantitative real-time PCR. The levels of P2X2 and P2X3 receptors mRNA were signifi cantly increased in diabetic mice at 14 days after the intravenous injection of STZ. Furthermore, we investigate the localization of glial cells and neuron in spinal cord of diabetic mice. However, microglia, astrocyte and neuron were not changed in spinal cord. These results suggest that the upregulation of P2X2, P2X3 and/or P2X2/3 receptor in DRG neurons is associated with mechanical allodynia in STZ-induced diabetic mice.
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