The hydrodynamic method by rapid intravenous injection of a large volume of plasmid DNA is known to be an efficient and liver-specific method of in vivo gene delivery and achieves high levels of foreign gene expression, particularly in hepatocytes. Low transgene activities have also been observed in other organs such as the spleen and lung; however, the expression profiles of mRNA and protein are still unknown. Therefore, we investigated the localization of luciferase mRNA by in situ hybridization and luciferase activity in mice after transfection of pCMV-luc encoding the luciferase gene under the control of cytomegalo virus (CMV) promoter. We found that hydrodynamic injection effectively induced mRNA expression of the transgene only in the liver although transgene activities were observed in other organs. The transgene activity observed in other organs may be due to leakage from hepatocyte gene expression by transient increase in the permeability of the hepatocyte cellular membrane caused by increased pressure by hydrodynamic injection.

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UVA-induced reactive oxygen species (ROS) generation is well known to cause cutaneous lesion in the skin, therefore, it is important to prevent the skin from UV damage due to ROS generation. In 2000, we reported first the in vivo detection and imaging by chemiluminescence (CL) method of the generated ROS in the skin of live mice following UVA-irradiation [1]. Using this method, we found that superoxide anion radical (•O₂¯) was intrinsically generated in the skin of live mice and singlet oxygen (¹O₂) was exclusively produced in the skin following UVA-irradiation. In our previously report, we found that topical application and oral administration of zinc compounds to the skin of live mice reduce the formation of UVA-induced ROS [2-3]. Then, in the present study, we examined whether metallogluconates (zinc gluconate (ZnGA) and copper gluconate (CuGA)) topically applied and orally administered reduce the intrinsic and UVA-induced ROS generations in the skin of live mice or not. We found that topical application and oral supplementation of CuGA reduced CL intensities in terms of the formation of UVA-induced ROS and ZnGA suppressed inflammation caused UVA-exposure.

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Previously we have described the development and applications of lipid-based nanoparticles for gene delivery vector. In an attempt to improve transfection efficiency using the cell adhesion of extracellular matrix (ECM) to DNA/lipid complex (nanoplex), the mRNA expression of integrin α2β1 and CD44 in prostate cancer cells was detected as adhesion molecules for fibronectin (Fn), collagen I (Col) and laminin (Lam) using a commercially available cDNA array (GEArray^TM) system. These ECM proteins could enhance DNA transfection activity in cells when coated on the nanoplex. Among the ECM proteins, Fn-coating nanoplexes significantly increased transfection activity 2-fold in prostate cancer PC-3 cells, and exhibited higher DNA transfection activities to PC-3 xenografts, compared with commercially available cationic polymer in vivo jetPEI. These results indicated that Fn-coating nanoplexes could facilitate efficient transfection of prostate tumor cells.

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Since it is proposed that reactive oxygen species (ROS) is involved in the pathogenesis of various diseases, low-molecular weight superoxide dismutase (SOD) mimetic complexes have been intensively studied. We prepared a Cu₂(aspirin)₄ complex consisting of Cu(II), active center of Cu, Zn-SOD, and aspirin, which has been in use for many years as an antipyretic, an analgestic, and as an anti-inflammatory agent. The ROS scavenging activities of Cu₂(aspirin)₄ and Cu(salicylic acid)₂, for comparison, have been evaluated in in vitro and in vivo. We have ever proposed that Cu₂(aspirin)₄ has SOD mimetic activity and defense activity against skin injury caused by an ultraviolet radiation. The suppressive effect of ROS generation following UVA irradiation on the skin of hairless mice, who received oral administration of the complexes for three consecutive days, was observed, the effect being significantly higher than that of Cu(II) ion. Further, the Cu concentration in the skin and blood of ICR mice who received complexes were measured using atomic absorption spectrometry. The Cu concentration in the skin of ICR mice treated with Cu₂(aspirin)₄ was slightly increased, probably due to lipophilicity of Cu₂(aspirin)₄ complex. The Cu₂(aspirin)₄ complex was thus observed to be an orally active anti-oxidative complex, proposing a potent anti-oxidative agent for clinical use in future to treat diseases relevant to ROS.

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Sodium lauryl sulfate (SLS), a representative anionic surfactant, is well-known to induce rough skin following single or multiple topical applications. The mechanism by which SLS induces rough skin is thought to result from the disruption of skin moisture function consisting of NMF and epidermal lipids. However, a recent study demonstrated that topically applied SLS easily penetrates into the living cell layers of the epidermis, which suggests that physiological alterations of keratinocytes might cause the SLS-induced rough skin. This study was conducted to clarify the effects of SLS on keratinocytes to demonstrate the contribution of SLS to the induction of rough skin. In addition, the potentials of other widely used anionic surfactants to induce rough skin were evaluated. HaCaT keratinocytes treated with SLS had increased levels of intracellular ROS and IL-1α secretion. Application of SLS on the surface of a reconstructed epidermal equivalent also showed the increased generation of ROS. Further, SLS-treated cells showed an increase of intracellular calpain activity associated with the increase of intracellular Ca²⁺ concentration. The increase of intracellular ROS was abolished by the addition of BAPTA-AM, a specific chelator of Ca²⁺. In addition, IL-1α also stimulated ROS generation by HaCaT keratinocytes. An ESR spin-labeling study demonstrated that SLS increased the fluidity of membranes of liposomes and cells. Together, those results indicate that SLS initially interacts with cell membranes, which results in the elevation of intracellular Ca²⁺ influx. Ca²⁺ stimulates the secretion of IL-1α due to the activation of calpain, and also increases ROS generation. IL-1α also stimulates ROS generation by HaCaT keratinocytes. We conclude from these results that the elevation of intracellular ROS levels is one of the causes of SLS-induced rough skin. Finally, among the other anionic surfactants tested, sodium lauryl phosphate has less potential to induce rough skin because of its lower generation of ROS.

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Nitric oxide (NO) from the endothelial NO synthase (eNOS) is believed to be implicated in the development and progression of atherosclerosis. The impaired endothelium-dependent vasodilatory response (EDR) has been demonstrated in vessels exposed to hypercholesterolemia and atherosclerosis. The extent of impairment serves as a predictor of future progression of atherosclerosis. As to the mechanisms of impaired EDR, increased production of superoxide is important. Recently it was revealed that eNOS becomes dysfunctional and produces superoxide rather than NO under conditions in which vascular tissue levels of tetrahydrobiopterin (BH₄), a co-factor for eNOS, are deficient or lacking. Dysfunctional eNOS is closely implicated in the endothelial dysfunction represented by impaired EDR in various vascular disorders including atherosclerosis. Regarding the role of eNOS in atherogenesis, experimental studies in vitro have revealed that NO from eNOS constitutes as an anti-atherogenic molecule. In eNOS-knockout mice, eNOS deficiency augments atherosclerotic lesion formation, although the effects may be partly due to the associated hypertension. However, in eNOS-transgenic mice (eNOS-Tg) crossbred with apolipoprotein E-deficient mice (apoE-KO/eNOS-Tg), we found the accelerated lesion formation in association with increased superoxide production from vessels compared with apoE-KO mice. The vascular tissue levels of BH₄ were reduced and BH₂, an oxidized form, levels were increased. Chronic administration of exogenous BH₄ or overexpression of GTPCH-1, a rate limiting enzyme for BH₄ synthesis, restored the lesion to the levels comparable to apoE-KO mice. Therefore, eNOS may have two faces in the pathophysiology of atherosclerosis depending on tissue BH₄ levels.

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Ascorbic acid (AsA) has multifunctional effects on skin beauty regarding the prevention and improvement of pigmentation and wrinkles. However, AsA not only has poor stability both in aqueous solutions and in cosmetic formulations, but it also influences the stabilities of formulations. To express the efficacy of AsA in/on the skin, it is important to develop a novel AsA derivative which overcomes those disadvantages. We synthesized 3-O-glyceryl ascorbate (3GA) by introducing a glycerol group into the C-3 position of AsA. 3GA has high stability in formulations. We then investigated the chemical and biological effects of 3GA. 3GA has a scavenging ability against various reactive oxygen species (ROS) assessed by the ESR spin-trapping method. HaCaT keratinocytes pretreated with 3GA have suppressed intracellular ROS levels both under physiological conditions and following exposure to H₂O₂ or UVB. In addition, HaCaT cells pretreated with 3GA were protected from cell damage induced by H₂O₂, UVB and ONOO^-. In reconstructed human epidermal equivalents, topical application of 3GA markedly reduced the generation of intracellular ROS after UVB irradiation. Taken together, 3GA is an effective AsA derivative that is stratum corneum permeable, does not interfere with the stability of cosmetic formulations, and is able to reduce intracellular ROS levels caused by oxidative stress.

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Recent increase of ultraviolet (UV) lights owing to the increase of ozone hole may induce enhancement of the skin damage in all living systems on the earth. The harmful effects of UV exposure on the skin are supposed to associate with the generation of reactive oxygen species (ROS), however, no direct proof of ROS production in the skin under UV exposure was established. Recently, we reported first the in vivo detection and imaging of ROS generation in the skin of live hairless animals following UV A exposure, using both the sensitive chemiluminescent probe (CLA) and the ultralow-light imaging apparatus with a CCD camera. It was proposed that ・O₂^- is formed spontaneously and ¹O₂ is generated by UV A exposure in the skin of live mice. The method was applied to find suppressive agents against skin damage by UV A exposure. The topical application of zinc(II) chloride suppressed the ROS generation induced by UV A exposure even after the removal of Zn(II) from the skin. The mechanism for suppression of skin damage by Zn(II) is now under investigation.

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バクテリアは，らせん形をしたべん毛繊維を回転させて水中を遊泳する．べん毛の働きは，スクリューに当たる．べん毛の正確な形状を観測することは，その微小なサイズと高速回転故に容易ではなかった．本論文では，レーザー暗視野顕微鏡を用いて，べん毛のらせんパラメータを計測する方法を提案し，サルモネラ菌へ適用した結果を示す．らせん形のべん毛を，らせん軸に対して45°または90°の方向からレーザービームで一方向照明すると，べん毛の像が一周期に1個または2個の輝点の列となる．そのような像を，電子シャッターを備えた高感度CCDカメラを使って記録した．記録した輝点の間隔を読み取ることで，菌体から脱落したべん毛（回転していない）のらせんピッチと半径を求めることができた．同様にして菌体上で高速回転するべん毛に関しても，ピッチの値が求まり，その微小な変化を捉えることもできた．

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To enhance tumor magnetic resonance imaging (MRI) signals via the selective accumulation of contrast agents, we prepared folate-modified gadolinium-lipid-based nanoparticles as MRI contrast agents. Folate-modified nanoparticles were comprised of polyethylene glycol (PEG)-lipid, gadolinium diethylenetriamine pentaacetic acid lipid, cationic cholesterol derivatives, folate-conjugated PEG-lipid, and Cy7-PEG-lipid. Folate receptor-mediated cellular nanoparticle association was examined in KB cells, which overexpress the folate receptor. The biodistribution of nanoparticles after their intravenous injection into KB tumor-bearing mice was measured. Mice were imaged through in vivo fluorescence imaging and MRI 24 h after nanoparticle injection, and the intensity enhancement of the tumor MRI signal was evaluated. Increased cellular association of folate-modified nanoparticles was inhibited by excess free folic acid, indicating that nanoparticle association was folate receptor-mediated. Irrespective of folate modification, the amount of nanoparticles in blood 24 h after injection was ca. 10% of the injected dose. Compared with non-modified nanoparticles, folate-modified nanoparticles exhibited significant accumulation in tumor tissues without altering other biodistribution, as well as enhanced tumor fluorescence and MRI signal intensity. The results support the feasibility of MRI- and in vivo fluorescence imaging-based tumor visualization using folate-modified nanoparticles and provide opportunities to develop folate targeting-based imaging applications.

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The use of radioisotopes and radiation is essential in the research field of life science. This article describes the studies on the usefulness of radioisotope in pharmaceutical sciences, especially focusing on the results of our research group as follows. (i) Mixed disulfide formation catalyzed by Cu (II) in relation to the radioprotective ability of a radioprotector, cysteamine (2-mercaptoethylamine). (ii) Suppression of reactive oxygen species (ROS) in the skin of live animals given oral administrations of Zn (II) and its complexes in relation to the skin damage under ultra-violet (UV) light. (iii) Improvement of diabetes mellitus by insulin-mimetic vanadyl (IV) (VO (II) ) complexes.

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一酸化窒素 (NO) は皮膚血流量の調節や血管の弛緩などに重要な役割を果たしていることが知られている。NOは生体内でNO合成酵素 (NOS) の作用によりL-arginine (L-Arg) から合成される。われわれはヒト血管内皮細胞において, (1) L-ピロリドンカルボン酸 (L-PCA) は濃度依存的に, NO産生量を促進する, (2) L-PCAは血管内皮細胞へのL-Argの取り込みを促進する, そして (3) この促進効果はカチオン性アミノ酸トランスポーター (CAT) 阻害剤であるL-NMMAによって抑制されることを見出した。これらの結果にもとついて, L-PCAはCATを介したL-Argの細胞への取り込みを促進することにより, NO生成量を適度に増加させ血流を調節していることがわかった。しかし, これらのNO合成促進およびL-Arg取り込み促進効果はL-PCAにのみ見られ, D-体には見られなかった。さらに, ヒト前腕内側部での閉塞パッチテストにおいても, L-PCAは皮膚の赤み・血流量を一時的に増加させることが認められた。これまでL-PCAは天然保湿因子の主要な成分の一つであることから保湿を目的としたスキンケアに広く用いられてきたが, 血流促進を目的とした用途においても有用であることが明らかとなった。

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Since it was proposed that reactive oxygen species (ROS) is involved in the pathogenesis of various diseases, superoxide dismutase (SOD) mimetic complexes have been intensively studied. We prepared a Cu₂(aspirin)₄ (Cu₂(asp)₄) consisting of Cu(II) and aspirin that has been in use for many years as an antipyretic, an analgesic, and as an anti-inflammatory agent. Also, Cu(salicylic acid)₂ (Cu(sal)₂) was used for comparison. The SOD mimetic activity was examined by using cytochrome c, electron spin resonance (ESR) spectroscopic and ESR spin trap methods; the activity of Cu₂(asp)₄ was almost equal to or less than those of Cu(sal)₂ and CuSO₄. The in vitro antioxidant activity was evaluated on the basis of the cell survival after ultraviolet B light (UVB) irradiation on an immortalized human epithelial/keratinocyte cell line (HaCaT) and normal human dermal fibroblast (NHDF). The cell survival ratio was found to significantly increase in the presence of Cu₂(asp)₄. Thus, the suppressive effect of ROS generation following UVA irradiation in the skin of hairless mice, who received oral administration of the compounds for three consecutive days, was found to be effective. The effect of Cu₂(asp)₄ was significantly higher than that of Cu(II) ion. Based on the results, Cu₂(asp)₄ complex was concluded to be a potent antioxidative compound in biological systems, and useful to treat diseases due to the involvement of ROS.

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Skin exposed to low humidity often shows hypersensitivity to sensory stimuli. The aim of this study was to identify the mechanism underlying such hypersensitivity to sensory stimuli using reconstructed epidermal equivalents (RHEEs) exposed to low humidity.

RHEEs exposed to low humidity were identified to be an appropriate model of dry skin by their secretion of interleukin (IL)-1α and carbonylated proteins (CPs). Oxidative situations and protein expression levels of nerve growth factor (NGF) and semaphorin3A (Sema3A) in RHEEs exposed to low humidity were compared with control RHEEs. It was found that RHEEs exposed to low humidity synthesized superoxide anion radicals (·O₂⁻) in a higher level. RHEEs exposed to low humidity also showed a higher level of NGF and a lower level of Sema3A compared to those of control RHEEs. In addition, treatment with the conditioned medium of RHEEs exposed to low humidity stimulated axon elongation in N1E-115 neuroblastoma cells. These phenomena were abolished by superoxide dismutase.

Thus, ·O₂⁻ generated in RHEEs exposed to low humidity caused an imbalance of NGF and Sema3A, which can result in creating a higher susceptible conditions against sensory stimuli.

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Our objective was to clarify the sleep states of bedridden elderly patients with disturbances of consciousness. We studied a bedridden elderly in patient with disturbances of consciousness for 24 hours in March 2010. We recorded the electroencephalograms, electro-oculograms, and electromyograms of the study subject. We classified the electrograms thus obtained into sleep stages according to international standards and identified various sleep variables and the patients' sleep structures. A patient showed very high values for sleep efficiency (SE), 97.8%. The number of awakenings was 36, the percentage of SWS was 1.0%, and the percentage of REM sleep was 8.5%. It is inappropriate to use SE and SWS as indicators of good sleep quality in such patients. To improve their sleep quality, further study is needed to determine better indicators of good sleep and to investigate respiratory conditions as a potential factor in sleep quality in this subgroup.

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We attempted to establish a dynamic diagnostic system of transplanted cells using a biochemiluminescence reaction that is catalyzed by luciferase. The cells used for transplantation were distributed from an established cell line that was collected originally from Wistar rats whose genome had systemic expression of the luciferase gene. The cultured cells retained their capacity for chemiluminescence, allowing us to use them in experimental transplantation. Neither replicative senescence nor stress-induced senescence was observed during passage of the cells. As the cells that were transplanted into the rat brains with an immunosuppressive agent showed sufficient viability, it was possible for us to identify the pathophysiology of the transplanted cells semiquantitatively by measuring the amount of chemiluminescence in vivo. The results of this study suggest that the luciferase-induced biochemiluminescence would be applicable to the establishment of new diagnostic and treatment techniques, and contribute to the development of a new field of imaging diagnostics.

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Gibberellins (GAs) are involved in many aspects of plant growth and development in higher plants. In this study, we aimed to characterize Satsuma mandarin (Citrus unshiu Marc.) GA 2-oxidase genes encoding enzymes with GA inactivation activity because the accumulation of active GAs is regulated by the balance between their synthesis and inactivation. We showed that CuGA2ox4, CuGA2ox2/3, and CuGA2ox8 were differentially expressed in various tissues in Satsuma mandarin and that these genes functioned like GA 2-oxidase genes in transgenic Arabidopsis. The expression pattern of CuGA2ox4 resembled that of CuGA20ox1, which was reported previously as a Satsuma mandarin GA 20-oxidase gene, in the reproductive tissues of the adult trees in that both genes were expressed in juice sacs in November and December and peel in December, suggesting that CuGA2ox4 and CuGA20ox1 act in concert to regulate the accumulation of active GAs in fruit at the maturing stage. On the other hand, CuGA2ox2/3 and CuGA2ox8 were more highly expressed in seeds and flower buds, respectively. Further study of GA biosynthetic genes, including GA 2-oxidase genes, would provide insight into the mechanism of flowering, fruit development, seedlessness, biennial bearing, and peel puffins of citrus such as Satsuma mandarin.

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The accumulation (incorporation) of paprika carotenoid in human plasma and erythrocytes was investigated. A paprika carotenoid supplement (14 mg/day) was ingested for 4 weeks by 5 young healthy volunteers (3 men and 2 women). After 2 weeks of carotenoid ingestion, the carotenoid levels in plasma and erythrocytes increased by 1.2-fold and 2.2-fold, respectively. Characteristic carotenoids found in paprika (capsanthin, cucurbitaxanthin A, and cryptocapsin) were detected in both plasma and erythrocytes. An oxidative metabolite of capsanthin (capsanthone) was also found in both plasma and erythrocytes.

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ウンシュウミカンは，2 つのジベレリン 20 酸化酵素遺伝子（CuGA20ox1 と CuGA20ox2）を有し，CuGA20ox1 のゲノム配列は CuGA20ox2 のそれより短く，CuGA20ox1 のコード領域は CuGA20ox2 のそれより長かった．サザンブロット解析により，他のカンキツ品種及びカラタチも少なくとも 2 つのタイプのジベレリン 20 酸化酵素遺伝子を有していることが示された．CuGA20ox1 と CuGA20ox2 の発現は組織において異なっており，CuGA20ox1 は生殖組織より栄養組織において比較的高い発現が見られたのに対し，CuGA20ox2 は開花直前の花雷に特異的に発現が見られた．これらの異なる発現様式は，進化の過程で上記 2 つの遺伝子の機能が分化したことを示唆している．CuGA20ox1 と CuGA20ox2 を異所的に発現した形質転換シロイヌナズナは花序が伸長したが，開花時期には変化が見られなかった．また，CuGA20ox1 と CuGA20ox2 の発現は，早期非水酸化経路上の GA₂₄ と GA₃₄ の産生に有意に影響を与えた．同様に早期水酸化経路上の GA₁₉，GA₂₉ 及び GA₈ の産生にも影響を与えた．これらの結果を考慮すると，CuGA20ox1 および/あるいは CuGA20ox2 は，早期水酸化経路及び早期非水酸化経路のどちらも活性化して活性型ジベレリンを増加させ，形質転換シロイヌナズナの花序伸長を引き起こしたと考えられる．

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