鈴木 敏和

【目的】 ビタミンDはカルシウム代謝に関わる脂溶性ビタミンとして知られている。近年、ビタミンDの不足者と心の健康の関連や、ビタミンD結合タンパク質をコードするgroup-specific component（GC）遺伝子の一塩基多型（SNP）と血中25-ヒドロキシビタミンDレベルの関連が報告された。そこで、心身的な健康状態やビタミンDサプリメントの心身的な健康状態改善効果にビタミンDの栄養状態が関連するか、さらにGC 遺伝子の一塩基多型とビタミンDサプリメントの心身的な健康状態改善効果の関連について見出すことを本研究の目的とした。 【方法】 本試験に同意した若年女性健常者76名を対象に、食物摂取頻度調査票を用いた栄養調査、外出時間や日よけ対策に関するアンケート調査、２か所（rs7041、rs4588）のGC遺伝子SNP型の調査、および４種の心身的な健康状態に関するアンケート調査（ベックの抑うつ質問票-II：BDI-II、特性不安尺度：STAI、知覚されたストレス尺度：PSS、Cook-Medleyの敵意スケール：Ho）を行った。その後90日間ビタミンDサプリメント（ビタミンD3として25μg/日）摂取させ、90日後に再び精神的な健康状態に関するアンケート調査を行った。 【結果】 栄養調査および外出時間や日よけ対策調査から試験参加者をビタミンD充足群と不足群に分けると、不足群において、心身的な健康状態のうち、BDI-IIのスコアが充足群と比較して1.5倍高かった。90日間のビタミンDサプリメント摂取により、不足群でBDI-IIスコアが有意に低下した。２か所のSNP型を合わせ、白人に多いSNP型所有者をグループ１、その他の者をグループ２と２群に分け、心身的な健康状態に関するアンケート調査の結果を比較したところ、サプリメント摂取前ではグループ１の方がグループ２よりもBDI-IIスコアが有意に高かった。また、ビタミンDサプリメント摂取によるBDI-IIスコア低下はグループ１でのみ観察された。 【考察】 若年女性において、ビタミンDが不足している人では抑うつ度が高まること、ビタミンDサプリメントによるうつ症状改善効果は、特にビタミンDが不足している人に有効である可能性が示唆された。また、グループ１の若年女性は抑うつ度が高く、ビタミンDサプリメント摂取によるうつ症状改善効果は、グループ２の者よりも高いことが示唆された。

The single nucleotide polymorphisms (SNPs) rs3808607, rs2072183, rs2032582, and rs1761667 are associated with coenzyme Q10 (CoQ10) bioavailability in women after long-term CoQ10 supplementation. However, the beneficial aspects of the association between these SNPs and CoQ10 supplementation remain unknown. We investigated their relationship using the subjective quality of life score SF-36 by reanalyzing previous data from 92 study participants who were receiving ubiquinol (a reduced form of CoQ10) supplementation for 1 year. Two-way repeated-measures analysis of variance revealed a significant interaction between rs1761667 and the SF-36 scores of role physical (p = 0.016) and mental health (p = 0.017) in women. Subgrouping of participants based on the above four SNPs revealed significant interactions between these SNPs and the SF-36 scores of general health (p = 0.045), role emotional (p = 0.008), and mental health (p = 0.019) and increased serum CoQ10 levels (p = 0.008), suggesting that the benefits of CoQ10 supplementation, especially in terms of psychological parameters, are genotype-dependent in women. However, significant interactions were not observed in men. Therefore, inclusion of SNP subgrouping information in clinical trials of CoQ10 supplementation may provide conclusive evidence supporting other beneficial health effects exerted by the association between these SNPs and CoQ10 on women.

【目的】CoQ10サプリメントの摂取は、アンチエイジング効果や健康維持への有用性が期待されている。しかし、同量のCoQ10サプリメントを摂取しても血清CoQ10レベルには個人差がみられ、この差がサプリメント効果の体感の差につながると考えられる。ビタミンやミネラル等の栄養素の吸収は、食事の影響を受けることから、CoQ10の吸収もまた食事の影響を受ける可能性が考えられる。本研究は、CoQ10サプリメント摂取臨床試験に参加している愛媛県上島町の住民を対象とし、CoQ10サプリメント長期摂取後の血清CoQ10レベルと食習慣との関連を検討した。【方法】愛媛県上島町の住民135名（男性43名：年齢61.9±12.7歳、女性92名：年齢62.6±12.4歳）を対象とし、CoQ10サプリメント(100～120mg/日)を摂取させた。サプリメント摂取前、および摂取開始から1年後に採血を行った。また、簡易型自記式食事歴法質問票を用いて食習慣を調査した。【結果】サプリメント摂取後に血中CoQ10レベルの高い被検者群では、サプリメント摂取前の血清CoQ10レベルも他の群と比較して高値を示した。食習慣においては、他の群と比較して男女ともに卵類や乳類の摂取が多い傾向にあった。【考察】CoQ10の体内への取り込みは、食習慣の影響を受ける可能性のあることが示唆された。また、乳化により安定なエマルションを形成し易い卵類や乳類といった食品、およびコレステロールを含んでいる食材とCoQ10を一緒に摂取することで、その吸収率が上がった可能性がある。サプリメントと同時に摂取する食品の組合わせを考慮することが、サプリメント効果体感率の改善につながると考えられた。

管理栄養士養成課程においては、ラットやマウスなどのモデル動物を用いた実験実習が行われている。動物実験は、栄養学や生化学、および解剖学など教科書で学んだ内容を短期間で体系的に学ぶことが出来るという利点と、生きた動物の命が犠牲になるという不利点が共存する。この不利点に視点が偏り、管理栄養士国家試験受験資格を得るために「仕方なく」実験実習に参加している学生も少なくない。本取り組みでは、実験動物の愛護に関する理念の3RのひとつであるReplacement、すなわち動物実験代替法を実習授業に組み込むことが、教育効果の改善につながるか検討を行った。

【目的】補酵素Q10（CoQ10）は、エネルギー産生や抗酸化能に関わる生命活動に必須の分子である。CoQ10は体内で生合成されるため、栄養学上は非必須栄養素に分類されている。目安となる摂取量は決められておらず、また食習慣と血中CoQ10値の関連を調査した報告もない。本研究では愛媛県上島町の住民を対象として、食習慣が血中CoQ10値に影響を与えているか、横断的調査による検討を行った。【方法】愛媛県上島町の住民189名（男性67名：年齢58.8±14.3歳、女性122名：年齢60.1±13.9歳）を対象とし、身体状況、血液・生化学的検査、栄養素摂取調査および生活習慣調査を実施した。【結果】血中CoQ10値は個人差が大きかった。食事からのCoQ10摂取量は、男女ともに60歳以上の方が多い傾向にあった。CoQ10を多く含む魚類および豆類の摂取量は、男女ともに60歳以上の方が有意に多かった。血中CoQ10値とCoQ10摂取量との間には明瞭な関連がみられなかった。【考察】血中CoQ10値は個人差が大きく、本横断的研究では食事による影響を見出すことはできなかった。一方、生合成経路が途中まで共通である血中総コレステロール値が、血中CoQ10値の高いグループでは低いグループよりも高かったことから、血中CoQ10値の個人差の一部は、生合成能力と関連がある可能性が示された。食習慣の影響を調べるには、横断研究における研究デザインの最適化やCoQ10摂取量を変化させるような食事介入調査を行うことが必要であると推測された。

本報告は、平成26年度和洋女子大学教育振興支援助成を受けて実施したものであり、健康栄養学類での管理栄養士育成に関わる教育において、「クリッカーシステム」を用いて教育効果を向上させ、学生の自己学習意欲を高めることを目的とした。クリッカーシステムは現在、本学「ワヨラ」で運用が行われているが、学生の講義への参加状況や理解度をリアルタイムで知ることができる。さらに学生参加型の双方向型授業によって授業においての集中力が高まること、自身の理解度を学友たちと相対比較することにより学習意欲向上が期待できる等の効果が言われている。 本取り組みでは、健康栄養学類の4年生に開講している国家試験対策関連講義で、クリッカーシステムを使用した授業を実施した。クリッカー使用スクリーンを可動式の第2スクリーンで適用することによって、授業運営をスムースにすることができた。学生アンケートの結果、授業の理解度を高める効果は確認できなかったが、クリッカーを用いた回答によって即座に学生の理解状況を確認できることで、学生の理解にあわせた授業を実施することができたとの評価を得た。クリッカーを通常授業で活用することに関しては、学生からはまだ積極的利用の要望が挙げられていないが、今後も引き続き、学習効果を高めるクリッカーの利用方法の検討をしていく必要があると考える。

The inhibitory effect of 13 taxanes isolated from the Chinese yew (Taxus chinensis var. mairei) on the proliferation of human cervical cancer HeLa cells were examined using an MTT assay. Four compounds having a hydrophobic cinnamate side chain showed antiproliferative activity, which may be due to increased cell permeability.

Objectives: Extracellular microenvironment plays crucial roles in the development of cancers and chemoresistance. Pancreatic carcinoma is resistant to almost all chemotherapeutic agents. In this study, we identified annexin II in the medium from pancreatic cancer cells as a protein released into the extracellular environment. Methods: Medium from 5-hour cultures of various cancer cells was collected. Proteins in the medium were detected by molecular mass analysis and immunoblotting. Anticancer drug sensitivity of cells preincubated with or without recombinant annexin II (rANX II) was measured using crystal violet assay and colony survival assay. Apoptosis- related molecules were analyzed by immunoblotting. Results: Recombinant ANX II supplementation in the medium confers resistance to anticancer drugs, including cisplatin, 5-fluorouracil, and gemcitabine, in MiaPaCa-2 and AsPC-1 cells. In MiaPaCa-2 cells, rANX II supplementation resulted in suppression of caspase-3 activation associated with increased Bcl-2/Bax ratios. Suppression of cisplatin-induced cell death by rANX II supplementation was canceled by inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase signal pathways. Conclusions: The current study is the first report to demonstrate that supplementation of rANX II in the medium increased resistance to anticancer drugs in pancreatic cancer cells. Recombinant ANX II exerts cell deathYsuppressive function by antagonizing cisplatin-induced apoptosis.

Kita, K., Sugita, K., Chen, S. P., Suzuki, T., Sugaya, S., Tanaka, T., Jin, Y. H., Satoh, T., Tong, X. B. and Suzuki, N. Extracellular Recombinant Annexin II Confers UVC-Radiation Resistance and Increases the Bcl-xL to Bax Protein Ratios in Human UVC-Radiation-Sensitive Cells. Radiat. Res. 176, 732-742 (2011). In this study, we found that refractoriness to ultraviolet (UVC) light-induced cell death was increased in UVC-radiation-sensitive cells derived from Cockayne syndrome patients when the cells were precultured in medium supplemented with recombinant annexin II (rANX II). In CS3BES cells, an immortal cell line derived from Cockayne syndrome patients, the rANX H supplementation-induced UVC-radiation resistance was suppressed by treatment with an anti-annexin II antibody and EGTA. The amount of biotinylated annexin II on the cell surface increased in the rANX II-supplemented cells but did not increase in the cells that were cotreated with rANX H and EGTA. The capacity to remove UVC-radiation-damaged DNA, (6-4) photoproducts and cyclobutane pyrimidine dimers, was the same in cells that were precultured with rANX II and in control cells that did not receive rANX II supplementation. The rANX II supplementation-induced UVC-radiation resistance was also observed in nucleotide excision repair-deficient cells and xeroderma pigmentosum group A-downregulated cells. The Bcl-xL to Bax protein ratios, an index of survival activity in cells exposed to lethal stresses, were increased in the cells that had been precultured in rANX II for 24 h prior to UVC irradiation. Treatment with a phosphatidylinositol 3-kinase inhibitor suppressed the increased UVC-radiation resistance and Bcl-xL to Bax ratios in the cells with rANX II supplementation. Furthermore, downregulation of Bcl-xL by siRNA transfection also suppressed the UVC-radiation resistance that was induced by rANX II supplementation. These results suggest that the increase in the Bcl-xL to Bax ratios may be associated with enhanced resistance to UVC-radiation-induced cell death. (C) 2011 by Radiation Research Society

Water was collected from the taps of houses in cities and from first-class rivers in eastern Japan, and organic compounds in the water were extracted and concentrated using an Oasis HLB 3-cc extraction cartridge. Human RSa cells were examined for assessing the cytotoxic effects of the concentrated compounds present in water by MTT assay. The viability of cells treated with tap water samples was found to be about 80-100Pompared with 100 168822344n cells treated with MilliQ water samples. The viability of cells treated with 17 river water samples was over 80 3.251414e+161xcept for the sample from the Aganogawa river; water samples from Tamagawa and Arakawa rivers showed less than 70% viability in their middle and lower sections. The deterioration in water quality was particularly evident in April and July in Tamagawa river and in August and October in Edogawa river. Tests of the cytotoxic effects of water samples using human RSa cells may be a more comprehensive approach in evaluating biological effects of various factors in tap and river water and in unifying the criteria that apply to environmental water quality.

Zahed M and Suzuki T contributed equally to the work.

Glucose-regulated protein 78 (GRP78) is expressed abundantly in various types of cancer cells and is believed to contribute to chemotherapeutic resistance. In this study, we investigated the effect of a continuous approach for the expression of a short hairpin RNA (shRNA) targeted to GRP78 with retrovirus transduction on the sensitivity to the anticancer drugs VP-16 and cisplatin. The reduction of GRP78 expression failed, and the expression of GRP94 and P5 chaperon mRNA increased; this increase was associated with a mild activation of the unfolded protein response in He La cells, which were stably transduced with GRP78 shRNA gene. The transduced cells exhibited similar sensitivity to VP-16-induced cell death when compared to control GFP shRNA gene-transduced cells. However, sensitivity to cisplatin-induced cell death was higher in GRP78 shRNA gene-transduced cells compared to control cells. These results demonstrate that the continuous or prolonged approach targeting GRP78 confers sensitization of He La cells to cisplatin independently of the down-regulation of GRP78 expression. The role of the unfolded protein response in sensitization to cisplatin is discussed.

We found increased expression of GRP94, the 94-kDa glucose-regulated protein, in HeLa cells 24h after treatment with luteolin. Luteolin increased the levels of GRP94 mRNA and protein, but it did not increase the expression of unfolded protein response (UPR)-regulated genes. In addition, luteolin also enhanced GRP94 promoter activity, suggesting that it enhances the expression of GRP94 at the transcriptional level, not via the UPR signaling pathway.

We screened SMG7-binding peptides with phage display and docking simulation analysis. Although a consensus motif was absent in the phage display-derived candidates, we succeeded to find a peptide CDDRPPKSC, which can bind specifically to SMG7. We conclude that docking simulation helps to find high-affinity peptides efficiently, even if the phage display-screened candidates lack a consensus region.

Delayed healing of skin wounds can be caused by wound instability, whereas appropriate massage or exercise prevents sclerosis and scar contracture. However, the mechanism by which wound healing is related to mechanical stress has not been fully elucidated. The present study aimed to identify whether mechanical stretching of fibroblasts reduces their production of extracellular matrix. We transferred skin fibroblasts into collagen-coated elastic silicone chambers, cultured them on a stretching apparatus, and used RT-PCR to examine the effects of mechanical stretching on the expression levels of 17 genes related to extracellular matrix production and growth factor secretion. We found that connective tissue growth factor (CTGF) was downregulated after 24 hr of cell stretching. Specifically, the CTGF mRNA and protein levels were 50% and 48% of the control levels, respectively. These findings suggest that cyclic stretching of fibroblasts contributes to anti-fibrotic processes by reducing CTGF production.

Corresponding Author

We investigated changes in the sub-cellular distribution of glycelaldehyde-3-phosphate dehydrogenase (GAPDH) after X-ray irradiation in HeLa cells. Twenty-four h after irradiation at 5Gy, nuclear GAPDH levels increased 2.6-fold, whereas total GAPDH levels increased only 1.2-fold. Knockdown of GAPDH using specific small interfering RNA (siRNA) led to sensitization to X-ray-induced cell death. These results suggest that GAPDH plays a role in the radioresponse.

To find novel proteins involved in radio-resistance of human cells, we searched for nuclear proteins, sional differential gel electrophoresis following mass spectrometry. We identified 6 proteins, whose levels were increased in nuclei 24 h after irradiation at 5 Gy, including aldolase A. Nuclear aldolase A levels increased twofold after the irradiation, however, total aldolase A levels did not change. When the expression of aldolase A was suppressed by its specific siRNA, sensitization of the suppressed cells to X-ray-induced cell death was observed. In addition, UVr-1 cells with higher aldolase A expression exhibited lower sensitivity to X-ray-induced cell death than the parental RSa cells with lower aldolase A expression. These results suggest that aldolase A may play a role in the radio-response of human cells, probably in nuclei, in addition to its glycolytic role in the cytosol. (c) 2008 Elsevier Inc. All rights reserved.

The 78-kDa glucose-regulated protein GRP78 is a central regulator of endoplasmic reticulum (ER) homeostasis, functioning in protein folding, ER calcium binding and modulation of transmembrane ER stress sensor activity. ER stress uncouples the interaction between GRP78 and ER stress sensors, leading to activation of the unfolded protein response (UPR), including upregulation of ER chaperone proteins. In the present study, we observed unexpected and remarkable induction of glucose-regulated protein 94 (GRP94) in HeLa cells following their transfection with 2'-O-methyl-modified siRNA specific to GRP78 mRNA. Additionally, we found that this siRNA also increased the expression of other UPR-induced genes, such as CHOP, ERdj4 and P5. Activation of UPR-dependent transcription and induction of apoptosis were also observed in cells transfected with GRP78 siRNA, Induction of apoptosis by GRP78 siRNA was also observed in PC-3 cells, which expressed high basal levels of GRP78 protein similar to that observed in HeLa cells. By contrast, five other human cell lines with lower basal expression of GRP78 protein did not undergo apoptosis when treated with GRP78 siRNA. Possible reasons for the strong activation of the UPR and apoptosis induced by GRP78 knockdown in HeLa cells, and the therapeutic utility of 2'-O-methyl-modified GRP78 siRNAs in anticancer treatment, are discussed. (C) 2007 Elsevier Inc. All rights reserved.

One of the most intriguing biological subjects is cell-surface molecules that regulate the susceptibility of human cells to cell-killing effects after irradiation with far-ultraviolet light (UV, principally 254 nm wavelength). Human RSa cells have unusual sensitivity to UV-induced cell-killing. We searched for molecules on the cell-surface of RSa cells that were present in different amounts as compared to a variant of these cells, UVr-1 cells, which have increased resistance to UV cell-killing. Among the 21 molecules examined, the amount of transferrin receptor (TfR) protein was found to be 2-fold higher in UVr-1 cells compared with in RSa cells. The amounts of this protein were also higher in the UV-resistant hematopoietic cell lines, CEM6 and Daudi, as compared to the UV-sensitive cell lines, Molt4 and 697. Culturing of UVr-1 cells in a medium containing anti-transferrin antibodies resulted in sensitization of the cells to UV cell-killing as demonstrated by colony formation assay. Similar results were observed by treatment of the cells with TfR siRNA. In contrast, overexpression of TfR protein led to a resistance to UV cell-killing in RSa cells and monkey COS7 cells as demonstrated by both colony formation and apoptosis assay. In TfR-overexpressing cells, reduction of p53 and Bax protein was observed after UV-irradiation. Thus, TfR expression appears to be involved in the regulation of UV-resistance, possibly via modulation of the amount of p53 and Bax protein.

Background. Little is known of the fibrinolytic host immune mechanisms responsible for induction of chronic allograft nephropathy (CAN), defined as a loss in glomerular filtration rate (GFR) caused by tubular atrophy and interstitial fibrosis, often with fibrous intimal thickening in the small arteries. However, chronic rejection has been reported to be associated with decreased activity of the fibrinolytic system. In our previous study, [Deamino-Cys(1), D-Arg(8)]-vasopressin (dDAVP) induced urokinase-type plasminogen activator (uPA) release from human peripheral T lymphocytes via arginine vasopressin (AVP) V-2-receptor-mediated reaction enhanced by an AVP V-1-receptor antagonist. Therefore, we examined the level of uPA released from peripheral T lymphocytes by AVP in transplant patients with CAN in comparison with control groups. Patients and Methods. In this study, we evaluated in vitro uPA releasing activity of lymphocytes obtained from renal allograft patients with well-functioning grafts (n = 9), CAN (n = 5), or acute rejection episodes (n = 5) compared with lymphocytes from healthy volunteers with normal renal function (n = 12) or patients with renal insufficiency (n = 5). Results. Lymphocytes prepared from patients with chronic allograft nephropathy showed a significantly lower increase in uPA release induced by the combination of the V-1-receptor antagonist and dDAVP compared with those from the other groups. Conclusion. This finding suggested that a decrease in uPA release from human peripheral blood lymphocytes by AVP-related peptides may be potentially involved in the pathophysiology of CAN.

Purpose: Radiation therapy is one of the standard treatments for cervical cancer. Glucose regulated protein 94 (GRP94) is a molecular chaperone, which increases in amount after X-ray irradiation. This study examined the involvement of GRP94 in radio-resistance in human cervical cancer cells. Materials and methods: Seven human cervical carcinoma cell lines (HeLa, SKG-I, SKG-IIIb, QG-U, Caski, SiHa and C33A) were examined for basal levels of GRP94 protein by western blotting analysis. Sensitivity to X-ray irradiation of these cell lines was determined with a colony survival assay. The suppression of GRP94 expression was performed using specific small-interfering RNA ( siRNA) in HeLa and Caski cells. Results: HeLa cells and QG-U cells, with higher basal levels of GRP94, exhibited a low sensitivity to X-ray cell killing. In HeLa cells, the sensitivity increased when protein GRP94 levels were reduced by specific siRNA transfection. However, a reduction in GRP94 protein had little effect on the X-ray sensitivity of Caski cells, which expressed low basal GRP94 protein levels but showed a low sensitivity to X-rays. Conclusions: High basal protein levels of GRP94 were correlated with a modest decrease in sensitivity to X-ray cell death in some cervical cancer cell lines. These results suggest that higher GRP94 protein expression is one of the molecular mechanisms causing resistance to radiation, and therefore GRP94 siRNA might be useful in tumor-specific gene therapy by reversing radio-resistance prior to radiation in cervical cancer.

Proteases have received attention as important cellular components responsible for stress response in human cells. However, little is known about the role of proteases in the early steps of cell response after X-ray irradiation. In the present study, we first searched for proteases whose activity levels are changed soon after X-ray irradiation in human RSa cells with a high sensitivity to X-ray cell-killing. RSa cells showed an increased level of fibrinolytic protease activity within 10 min after irradiation with X-ray (up to 3 Gy). The induced protease activity was proved to be inhibited by leupeptin. We next examined whether this protease inducibility is related to the X-ray susceptibility of cells. Treatment of RSa cells with leupeptin prior to X-ray irradiation resulted in lowered colony survival and an increased ratio of G(2)/M-arrested cells and apoptotic cells. These results suggest that leupeptin-sensitive proteases are involved in the resistance of human RSa cells to X-ray cell-killing. (c) 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

[deamino-Cys(1), D-Arg(8)]-vasopressin (dDAVP), known to be an arginine vasopressin (AVP) V-2 receptor agonist, is an agent that increases fibrinolytic activity levels in plasma after its infusion into the human body. However, mechanisms underlying an increase and exact localization of the extrarenal dDAVP-responsiveV(2) receptor remain unclarified. Two AVP receptors, V-1a and V-2, and a related oxytocin (OT) receptor were found to be expressed in human lymphocytes. Furthermore, we found an increase of fibrinolytic activity in the medium of peripheral lymphocytes obtained from human volunteers less than 20 min after dDAVP infusion. The increased activity was also detected in the medium after incubating the lymphocytes in the presence of dDAVP in vitro, being highest at 20 min after the incubation. In accord with the increased fibrinolytic activity, the levels of urokinase-type plasminogen activator (uPA) in the medium were also increased. However, there was no significant difference of plasminogen activator inhibitor-1 (PAI-1), pro-uPA, and tissue-type plasminogen activator (tPA) concentrations in the medium between dDAVP treatment and control. When lymphocytes were preincubated with a V-2 receptor antagonist [Adamantaneacetyl(1), O-Et-D-Tyr(2), Val(4), Aminobutyryl(6), Arg(8,9)]-vasopressin, the dDAVP-induced uPA increase was diminished. In contrast, preincubation with a V-1 receptor antagonist, [beta-Mercapto-beta,beta-cyclopentamethylenepropionyl(1),O-Me-Tyr(2), Arg(8)]vasopressin, prior to dDAVP treatment resulted in a greater increase of the uPA concentration in the medium than with the dDAVP treatment alone. Thus it was suggested that dDAVP may induce uPA release from human lymphocytes via V-2 receptor-mediated reaction, and also via cross-talk between V-1 and V-2 receptors.

The relationship between oxidative stress and longevity is a matter of concern in various organisms. We isolated mutants resistant to paraquat from nematode Caenorhabditis elegans. One mutant named mev-4 was long-lived and showed cross-resistance to heat and Dyf phenotype (defective in dye filling). Genetic and sequence analysis revealed that mev-4 had a nonsense mutation on the che-11 gene, homologues of which are involved in formation of cilia and flagella in other organisms. The paraquat resistance was commonly observed in various Dyf mutants and did not depend on the daf-16 gene, whereas the extension of life span did depend on it. Expression of antioxidant enzyme genes seemed normal. These results suggest that chemosensory neurons are a target of oxidative stress and influence longevity dependent on the daf-16 signaling in C. elegans.

5-Bromodeoxyuridine (BrdU) induces a phenomenon similar to cellular senescence in mammalian cells. To address an underlying molecular mechanism in this phenomenon, we assessed the role of AT-hook proteins that bind to the minor grooves of specific AT-rich sequences. We expressed DsRed-tagged HMGI, MATH2, and MATH20 proteins in HeLa cells in a doxycycline dependent manner. Modest expression of these proteins revealed no apparent effect on the cells although high levels of expression were toxic to the cells. In contrast, their modest expression in the presence of low concentrations of BrdU similarly and dose-dependently induced senescence markers examined, although the same concentrations of BrdU alone showed no obvious effect. In both cases, DsRed fluorescence was mainly observed as foci or intense dots on Hoechst 33342-staining regions. These distribution patterns were not changed by addition of BrdU. Since AT-hook domains can displace chromatin compacting proteins pre-bound on AT-rich sequences, these results suggest that chromatin unpacking is one of the factors stimulating expression of the senescence markers in human cells. (C) 2003 Elsevier Inc. All rights reserved.

5-Bromodeoxyuridine (BrdU) immediately and clearly suppresses expression of the mouse Myod1 and human MYOD1 genes in myoblastic cells. Despite various studies, its molecular mechanism remains unknown. We failed to identify a BrdU-responsive element of the genes in experiments in which reporter constructs containing known regulatory sequences were transferred to mouse C2C12 myoblasts. Therefore, we transferred human chromosome 11 containing the MYOD1 gene to the cells by microcell-mediated chromosome transfer. In the resulting microcell hybrids, BrdU suppressed expression of the transgene, as determined by quantitative real-time RT-PCR analysis. We then transfected human PAC clones containing the MYOD1 gene to the cells. In the resulting transfectants, BrdU suppressed the transgene similarly. Deletion analysis suggested that a BrdU-responsive element or chromatin structure exists between 24 and 47 kb upstream of the gene. These results are the first demonstrating BrdU-responsiveness of a transgene for the known BrdU-responsive genes and facilitating determination of its precise responsible structure.

5-Chlorodeoxyuridine (CldU) immediately induces a senescence-like phenomenon in any type of mammalian cells probably due to a change in nuclear matrix structure. We thus examined nuclear matrix proteins in HeLa cells cultured with CIdU by high-resolution two-dimensional gel electrophoresis and peptide mass spectrometry. Three proteins were found to be down-regulated and one protein up-regulated by addition of CIdU. In addition, one acidic protein accumulated in the nuclear matrix, although not quantitatively changed in the nuclei. Since these alterations were observed within 24 It after addition of CIdU, these proteins may be involved in an early step of the senescence-like phenomenon. (C) 2002 Elsevier Science Inc. All rights reserved.