Measurement of intracellular pH by flow cytometry using pH sensitive fluorescence dye, and influence of hyperthermia and amiloride derivatives on the intracellular pH

エールリッヒ腹水癌細胞とそのアドリアマイシン耐性細胞において蛍光pH指示薬2'、7'-bis-(2-carboxyethyl) carboxyfluorescein] (BCECF) の蛍光量をフローサイトメトリーで測定することによって細胞内pHの検量曲線を作成することができた。このことより、これらの細胞においてBCECFの蛍光量で細胞内pHの変化を簡易に比較できることを示唆した。さらに、温熱、Na(+)/H(+) exchanger の阻害例であるアミロライド[3,5-diamino-6-chloro-N-(diaminomethylene) pyrazinecarboxamide]、およびアミロライド誘導隊MH-12-43[N-amidino-3-amino-6-chloro-5-(N-ethyliso-propylamino) pyrazinecarboxyamide] の細胞内pHへの影響をエールリッヒ腹水癌細胞で観察した。37℃では、0.5mMアミロライド、0.05mM　MH-12-43により細胞内pHは減少し、42℃処理によりさらに減少した。42℃において、0.05mM　MH-12-43による細胞内pHの減少は、0.5mMアミロライドによる減少より大きかった。We examined relationship between intensity of intracellular fluorescence of [2', 7'-bis-(2'-carboxyethyl) carboxyfluorescein] (BCECF) and intracellular pH in Ehrlich ascites tumor cells and their adriamycin-resistant strain, and found a good correlation between them at both strains. This suggests that changes in the intracellular pH on these strains may be obtained through measurement of intracellular fluorescence of BCECF by flow cytometry. Further, we examined influence of hyperthermia, 3, 5-diamino-6-chloro-N-(diaminomethylene)pyrazinecarboxamide (amiloride), an inhibitor of Na(+)/H(+) exchanger, and its derivative; N-amidino-3-amino-6-chloro-5-(N-ethylisopropylamino) pyrazinecarboxyamide (MH-12-43) on the intracellular pH in Ehrlich ascites tumor cells. The treatment of 0.5mM amiloride or 0.05mM MH-12-43 reduced intracellular pH at 37℃, while the more reduction was observed by the treatment at 42℃. The reduction of intracellular pH by 0.05mM MH-12-43 was more substantial than that of 0.5mM amiloride at 42℃

Cepharanthin Reduces Thermotolerance by Enhancing Thermosensitivity in NIH3T3 Cells

The effects of cepharanthin (Ce), glycyrrhizin (G), verapamil (V), and G plus V on induced thermotolerance in NIH3T3 cells were studied. Cells were heated with or without the drug at 45 degrees C for 20 min (the first heating), incubated at 37 degrees C for 12h (the incubation period), and heated again at 45 degrees C for 0-210 min (the second heating). G and V were added throughout the experiment, while Ce was added throughout the experiment or during only the first or second heating, or the incubation period. The cells were harvested after the second heating to evaluate cell survival. In control experiments without any drug, thermotolerance developed and reached the highest peak in the cells incubated for 12h at 37 degrees C. However, thermotolerance in the control cells was suppressed by incubating them at 0 degree C, but developed by subsequent incubation at 37 degrees C. This suggests that the acquisition of thermotolerance by the cells required metabolic processes during the incubation at 37 degrees C. When each drug was present throughout the experiment, only Ce or the combined use of G and V was effective in reducing thermotolerance. Thermotolerance was also suppressed in the presence of Ce during the second heating. These results indicate that Ce reduces thermotolerance by enhancing thermosensitivity rather than by inhibiting the development of thermotolerance.</p

Development of Bioactivity and Pull-out Torque Control Technology on Ti Implant Surface and its Application for Cold Thread Rolled Bone Screw

AbstractThe influence of cold thread rolling conditions and a production method of titania (TiO2) films on surface bioactivity and pull-out torque of a titanium bone screw was investigated. The bone screw with micro surface roughness distribution was formed by cold thread rolling with a pair of parallel dies. The die shape and surface roughness distribution were changed to the 3 grades. The TiO2 films were coated on the surface of the bone screw using anodizing in aqueous solutions (hydro-coating). We introduced the rolled bone screws into tibia of rats for two weeks and examined the effects of the combinations of the surface morphology and the TiO2 film on osteoconductivity in an in-vivo experiment. As the results, it is found that we could control the bioactivity and pull-out torque by controlling the surface roughness at the bottom of the screw root

加温後のtsAF8細胞の細胞周期

Thermotolerance in tsAF8 cells develops during incubation at 34℃ after heating at 45℃, while it is suppressed by the following incubation at a non-permissive temperature of 39.7℃ after the same heating. The incubation temperature after heating may affect the cell cycle and consequently thermotolerance. In the present study, a relationship between the thermotolerance and the cell cycle of tsAF8 was investigated. The cell cycle fractions and DNA synthesis were measured by flow cytometry using double staining with propidium iodide and bromodeoxyuridine. When the tsAF8 cells were heated at 45℃ for 20 min, and thereafter incubated at 34℃, bromodeoxyuridine uptake in the S phase cells (DNA synthesis) was recovered to 65.1% 6 h after the heating, and the cells showed gradual accumulation in the G(2)/M phase. When the cells were incubated at 39.7℃ after heating at 45℃ for 20 min, then showed inhibition of thermotolerance development, the DNA synthesis was recovered to 15.1% temporarily 6 h after the heating, but it became 0% after 12 h, and the cells did not remarkably accumulate in any phases of the cell cycle. This inhibition of DNA synthesis at 39.7℃ was considered to be the result of cell survival decreasing by a step-down heating. However, the relationship between the thermotolerance and the cell cycle was not found out in tsAF8 cells, because the cells did not accumulate in any phases of the cell cycle under the inhibitory condition of thermotolerance.tsAF8細胞は45℃の加温後34℃で培養すると温熱耐性が速やかに発現するが,加温後,制限温度である39.7℃で培養すると温熱耐性の発現が抑制される｡加温後の培養温度が細胞周期に影響し,その結果として温熱耐性発現に影響を与えている可能性があることから,今回,Propidium Iodide(PI)とbromodeoxyuridine(BrdU)でtsAF8細胞を二重染色し,フローサイトメトリーによって温熱耐性と細胞周期の関係の有無について調べた｡tsAF8細胞を45℃20分の加温後34℃で培養すると,6時間後にはG(1)期の細胞が減少し,12時間後にはG(2)/M期への蓄積が見られた｡しかし,加温後39.7℃で培養した場合には細胞周期の進行がほとんど見られなかった｡BrdU の取込みは,加温せずに39.7℃で培養した場合には活発に行われ,また,45℃20分加温後34℃で培養した場合には,6時間後にはBrdUの取り込みは65.1%まで回復した｡しかし,温熱耐性発現の抑制が観察される45℃20分加温後39.7℃で培養した場合には,BrdUの取込み量は6時間後に一時的に15.1%に回復するが,12時間後には取込み量はゼロとなった｡BrdUの取り込みが阻害されたのはstep-down heatingの現象による細胞生存率の減少が原因だと考えられたが,温熱耐性発現の抑制が観察される条件下では細胞周期の特定の時期への集積がなかったことから,温熱耐性と細胞周期との関係はtsAF8細胞においては見い出されなかった

Relationship between intracellular uptake of adriamycin and membrane potential in ADR resistant Ehrlich ascites tumor cells

We observed adiamycin (ADR) uptake and cellular transmembrane potential [amount of intracellular fluorescence of 3,3'- (Di-n-hexyl)- 2,2'- oxacarbocyanine iodide (NK-2280)] in ADR-resistant cells established from Ehrlich ascites tumor cells (EATC) and wild type EATC. In ADR-resistant cells, ADR uptake and the cellular transmembrane potential decreased as the degree of resistance increased. 4,4'- diisothiocyanatostilbene- 2,2'- disulfonic acid (DIDS) induced markedly decreases of ADR uptake and the cellular transmembrane potential. A good correlation was observed between ADR uptake and transmembrane potential in cultured cells

Conduction Effect of Thermal Radiation in a Metal Shield Pipe in a Cryostat for a Cryogenic Interferometric Gravitational Wave Detector

A large heat load caused by thermal radiation through a metal shield pipe was observed in a cooling test of a cryostat for a prototype of a cryogenic interferometric gravitational wave detector. The heat load was approximately 1000 times larger than the value calculated by the Stefan-Boltzmann law. We studied this phenomenon by simulation and experiment and found that it was caused by the conduction of thermal radiation in a metal shield pipe.Comment: 7 pages, 4 figures, 2 tables, Submitted to Jpn. J. Appl. Phy

Application of hot spring pool training in outpatients with chronic diseases and its clinical evaluation

1996年4月より6月までの3ヵ月間に,三朝分院内の温泉プールで水泳ないし歩行訓練を受けた内科外来患者は延べ2109例(実数213例)で,外来患者総数(延べ6848例)に対する利用率は30.8%であった｡これらの対象症例について,リハビリテーション受診カードにより,性別,年令別,疾患別の検討を行った｡その結果,性別では女性が多く(72.3%),年齢別では50才以上の患者が多い(80.3%)傾向が見られた｡また,地域別検討では,鳥取県内からの患 者が多い(88.3%)傾向であった。さらに,1996年12月より1997年1月へかけて,アンケート調査を行い(集計数;64例)継続期間,利用回数,効果を感じた期間,効果のあった症状などについて疾患別に比較検討した｡その結果,慢性閉塞性肺疾患を主とした呼吸器疾患では,週に1～2回の利用回数(50.0%)で長期間(3年以上:52.9%)続けている患者が多い傾向であった｡また慢性関節リウマチ,腰痛などの慢性疼痛性疾患では,訓練期間は比較的短期間であっ たが（3年以下:52.9%),利用回数は週に3～5回(38.0%)と多い傾向が見られた。温泉プール訓練では,疾患本来の症状改善に加えて,全身状態の改善,精神的リラックス,気分転換などを感じていることが示唆された｡For three months from April to June in 1996, 2109(30.8%) of 6848 outpatients (the total number) had swimming and walking traning in a hot spring pool. Of the 213 patients (the actual number), the numbers of female patients (72.3%), of patients over the age of 50 (80.3%), and of those coming inside Tottori prefecture (88.3%) were larger in analysis by the consultation cards for rehabillitation. The results of questionnaire from the patients performed from December 1996 to January 1997 demonstrated that patients with chronic respiratory disease had pool training 1 to 2 times a week (50.0%) and for longer term (more than 3 years, 52.9%), and patients with chronic pain disease had the training 3 to 5 times a week (38.0%) and for shoter term (less than 3 years, 61.9%). It was suggested from the results that tha training in a hot spring pool improved subjective and objective symptoms of each disease, and, furthermore, brought about improvement of general condition, psychological relaxation, and a change of feeling

A new capacitive heating applicator for the simultaneous radiohyperthermotherapy of superficial and shallow-seated tumors.

External capacitive heating is the usual method of electromagnetic wave heating, in which the tumor is caught and heated between two opposite applicators. Using a phantom, the authors developed and evaluated the performance of a new capacitive heating applicator designed for simultaneous radiohyperthermotherapy (SRH) in which the electron beam irradiation is provided from above an external capacitive heating applicator for the treatment of superficial and shallow-seated tumors. The trial applicator was constructed to fulfill the following conditions: 1. use of an electrode plate which does not affect the electron beam depth dose, 2. a uniform thickness to maintain flatness of the electron beam, and 3. a cooling function to prevent damage to normal skin tissue and enhance the therapeutic gain factor. This applicator was comprised of a 0.1-mm-thick copper electrode and a 5-mm-thick cooling chamber. The depth of the 80% dose of the new applicator was 21 mm with a 9-MeV electron beam and 36mm with a 15-MeV electron beam, which was comparable to the effect of a conventional irradiation bolus. The temperature distribution produced by the trial applicator was symmetrical on both sides from the center of the applicator. The 50% specific absorption rate region was 6.4 cm wide at a depth of 1 cm from the phantom surface and 2.8 cm wide at a depth of 3 cm. There have been no previous reports on the development of an external capacitive heating applicator designed for the SRH of superficial and shallow-seated tumors; this is the first such report.(ABSTRACT TRUNCATED AT 250 WORDS)</p

The Results of Radiotherapy for T1 Glottic Cancers: Influence of Radiation Beam Energy

We analyzed the influence of various parameters on the results of radiotherapy for T1 glottic cancer by assessing the outcomes of 60 patients with this cancer who received definitive radiotherapy between 1985 and 1994. Seven patients were treated with a cobalt-60 unit, and the other 53 with a linear accelerator (26 patients at 3-MV, 10 at 6-MV, and 17 at 10-MV). Of the 17 patients treated at 10-MV, 4 also received part of their treatment with a cobalt-60 unit. The total radiation dose ranged from 56 Gy to 70 Gy (mean, 61 Gy). The total radiation dose of 51 patients (85%) was 60 Gy. The factors found to influence local control were the strength of the radiation beam energy and whether or not there was gross tumor invasion of the anterior commissure. The local control rate was 71% in the patients treated with a 10-MV linear accelerator, 56% in those treated with a 6-MV linear accelerator and, 97% in those treated with a cobalt-60 unit or a 3-MV linear accelerator (P = 0.0173). The local control rate was 43% in the patients with gross anterior commissure invasion and 88% in those without (P = 0.0075). We conclude that low energy photon beams are more suitable for the treatment of early glottic cancers, especially if the lesion grossly invades the anterior commissure