Nuclear Factor (Erythroid-Derived)-Related Factor 2-Associated Retinal Pigment Epithelial Cell Protection under Blue Light-Induced Oxidative Stress

Purpose. It is a matter of increasing concern that exposure to light-emitting diodes (LED), particularly blue light (BL), damages retinal cells. This study aimed to investigate the retinal pigment epithelium (RPE) damage caused by BL and to elucidate the role of nuclear factor (erythroid-derived)-related factor 2 (Nrf2) in the pathogenesis of BL-induced RPE damage. Methods. ARPE-19, a human RPE cell line, and mouse primary RPE cells from wild-type and Nrf2 knockout (Nrf2−/−) mice were cultured under blue LED exposure (intermediate wavelength, 450 nm). Cell death rate and reactive oxygen species (ROS) generation were measured. TUNEL staining was performed to detect apoptosis. Real-time polymerase chain reaction was performed on NRF2 mRNA, and western blotting was performed to detect Nrf2 proteins in the nucleus or cytoplasm of RPE cells. Results. BL exposure increased cell death rate and ROS generation in ARPE-19 cells in a time-dependent manner; cell death was caused by apoptosis. Moreover, BL exposure induced NRF2 mRNA upregulation and Nrf2 nuclear translocation in RPE. Cell death rate was significantly higher in RPE cells from Nrf2−/− mice than from wild-type mice. Conclusions. The Nrf2 pathway plays an important role in protecting RPE cells against BL-induced oxidative stress

体外培養Babesia gibsoniにおけるジミナゼン耐性に関する研究

Canine babesiosis caused by Babesia gibsoni is treated with diminazene aceturate (DA). DA can temporarily improve the clinical signs of canine babesiosis but is unable to eliminate the parasites from infected dogs, and relapses often occur. Therefore, it is believed that B. gibsoni might achieve the resistance against DA. However, there is no report clearly demonstrating the DA resistance in B. gibsoni. Therefore, in this study, a DA-resistant B. gibsoni strain was developed and the differences between the DA-resistant B. gibsoni strain and wild-type B. gibsoni were compared. First, a DA-resistant B. gibsoni strain was developed in vitro by the gradual increase of the DA concentration from 1 to 200 ng/ml. The parasites survived and proliferated in the medium containing 200 ng/ml DA, which is much higher than the 50% inhibitory concentration (IC50) of DA for B. gibsoni. Subsequently, these parasites were removed from erythrocytes and exposed directly to 200 ng/ml DA. They survived and invaded fresh erythrocytes, though wild-type B. gibsoni did not survive. Based on these results, the parasites cultured with 200 ng/ml DA were determined as a DA-resistant B. gibsoni strain. Thereafter, to investigate the characteristics of the DA-resistant B. gibsoni strain, the effects of other antibabesial drugs, including clindamycin, doxycycline, metronidazole and pentamidine, on the DA-resistant B. gibsoni strain were examined. The DA-resistant B. gibsoni strain showed strong resistance against pentamidine, and weak resistance against clindamycin and doxycycline. Moreover, the IC50 values of clindamycin, doxycycline and pentamidine for the DA-resistant strain at day 7 were higher than those for the wild-type B. gibsoni, respectively. These results indicated that the DA-resistant B. gibsoni strain could have resistance not only to DA, but also to other antibabesial drugs. Especially the DA-resistant B. gibsoni strain exhibited resistance against pentamidine, which shares similar structure with DA. In other protozoan, the mechanisms of drug resistance through mutations and/or amplification in drug transporters or drug targets were demonstrated. Therefore, the analysis for those metabolic pathways in the DA-resistant B. gibsoni strain will lead to elucidate the mechanism of the action of DA against B. gibsoni.Consequently, to characterize the DA-resistant B. gibsoni strain, the transcription levels of B. gibsoni heat shock protein 70 (BgHsp70) gene, which plays important roles in cell proliferation and the control of cellular function, was measured by quantitative real-time reverse transcription-polymerase chain reaction. In Plasmodium falciparum, Hsp70 has been proposed to contribute to the development of drug resistance. Therefore, the change in the transcription levels of the BgHsp70 gene was analyzed in DA resistance. During the development of the DA-resistant B. gibsoni strain, DA-resistant B. gibsoni variants, which were maintained in culture with DA from 1 to 175 ng/ml for more than 8 weeks, were also obtained. The copy number of the BgHsp70 transcripts in the DA-resistant variant cultured with 1 ng/ml DA was significantly lower than in wild-type B. gibsoni while those in DA-resistant variants increased with escalating doses of DA from 1 to 75 ng/ml, though they were lower than in wild-type B. gibsoni. Moreover, those in DA-resistant variants cultured with > 125 ng/ml DA were almost the same as wild-type B. gibsoni. It is hypothesized that the transcription levels of the BgHsp70 gene would be reduced during the selection of the DA-resistant B. gibsoni strain under the long-term weak pressure of DA, and then would be returned to the normal level after achieving resistance against DA. However, since the reason why the transcription levels of the BgHsp70 gene was reduced is still unclear, further study will be necessary to confirm this hypothesis. In conclusion, it was clearly demonstrated the development of DA resistance of B. gibsoni in vitro. The DA-resistant B. gibsoni strain obtained resistance against other antibabesial drugs. Moreover, the transcription levels of the BgHsp70 gene was reduced by the weak DA pressure and then recovered when B. gibsoni had achieved resistance against DA. However, the role of BgHsp70 for the DA resistance in B. gibsoni remains unclear. Further studies of BgHsp70 might prove to determine the mechanism of the DA resistance of B. gibsoni. Finally, the results obtained from this study could contribute to a better understanding of the DA resistance in B. gibsoni in vitro.Babesia gibsoniにより引き起こされる犬バベシア症は抗バベシア原虫薬の酢酸ジミナゼン(diminazene aceturate, DA)の投与により治療するが、原虫を犬体内から完全に排除することは困難であり、再発を繰り返すとされている。この原因として、原虫がDA耐性を獲得することが疑われているが、今まで実験的にDA耐性株の作製を行った例や、その解析を行った報告はない。そこで本研究では、DAに対して薬剤耐性を示すB. gibsoniの作製を試み、その耐性株の特性を解析した。初めに、培養にて維持しているB. gibsoniを用いて、DA耐性B. gibsoni株の作製を行った。このために、培養液に含まれるDA濃度を1 ng/mlから開始して除々に増加させ、最終的に200 ng/mlまで増加させた。さらに、200 ng/mlのDAを含んだ培養液中で維持している原虫を感染赤血球から分離して200 ng/mlのDAを含む培養液に直接曝露し、原虫の赤血球への再侵入と増殖を観察した。その結果、DAに耐性を持たないB. gibsoni (野生株)は、同様の実験で生存できなかったが、200 ng/mlのDAで維持している原虫は、新しい赤血球に侵入し、増殖した。B. gibsoniに対するDAの50%の阻害濃度(IC50)は45.89 ng/mlあるいは103 ng/mlと報告されているため、これはDA耐性株であると考えられた。以上より、このB. gibsoniはDAに対する耐性を獲得したと考えられた。次に、このDA耐性B. gibsoni株の特徴を明らかにするため、様々な種類の作用機序を持つ抗バベシア原虫薬に対するDA耐性B. gibsoni株の抵抗性を比較した。すなわち、作製したDA耐性B. gibsoni株及び野生株をクリンダマイシン、ドキシサイクリン、メトロニダゾ－ル及びペンタミジンをそれぞれ含む培養液中にて7日間培養し、各薬剤に対する抵抗性を比較した。その結果、DA耐性B. gibsoni株はクリンダマイシン、ドキシサイクリンに対して弱い抵抗力を示し、ペンタミジンに対して強い抵抗性を示した。特にDAと類似の作用機序を持つペンタミジンに対しては高い抵抗性を示し、同系統の薬剤に対して強い耐性を示すと考えられた。さらに、それぞれの薬剤に対するDA耐性B. gibsoni株のIC50は上昇した。よって、以上の薬剤の作用する代謝経路を比較することで、DAのB. gibsoniに対する作用機序や、DA耐性の機序が明らかになることが期待できる。さらに、このDA耐性B. gibsoni株の特徴を明らかにするため、細胞機能に重要な役割を果たすとされている熱ショックタンパク質70 (heat shock protein 70, Hsp70)遺伝子の転写量を定量的リアルタイムPCR法(quantitative real-time reverse transcription-polymerase chain reaction; qRT-PCR)で測定した。Plasmodium falciparumにおいてHsp70は薬剤耐性の獲得に貢献しているとの報告があり、B. gibsoniのHsp70 (BgHsp70)もDA耐性の獲得に貢献していることが期待された。DA耐性B. gibsoni株を作製する過程で、1 ng/mlから175 ng/mlまでのDA存在下で8週間以上維持しているDA耐性B. gibsoni変異株を分離した。これらの変異株のBgHsp70遺伝子の転写量を測定したところ、1 ng/mlのDA存在下で維持している変異株のBgHsp70遺伝子転写量は野生株に比べて有意に低く、DA濃度が1 ng/ml から75 ng/mlまで増加するにつれてBgHsp70遺伝子の転写量が上昇した。さらに、培養液中のDA濃度が125 ng/ml以上で維持している変異株では、BgHsp70遺伝子の転写量が野生株と同程度であった。以上の結果より、DA耐性B. gibsoni変異株のBgHsp70遺伝子の転写量は低濃度でのDAに長時間曝されることで減少し、DA耐性を獲得した後に野生株と同程度に回復することが推測された。しかしながら、BgHsp70遺伝子の転写量が減少した原因はまだ解明されていない。今後、BgHsp70の機能を明らかにすることで、DA耐性の機序が明らかになるかもしれない。本研究では、DA耐性B. gibsoni株の作製に成功した。このDA耐性B. gibsoni株は、DAのみならず、他の抗バベシア原虫薬に対する抵抗性も増加していた。さらに、低濃度のDAにより、BgHsp70遺伝子の転写量が減少することも明らかになった。しかしながら、DA耐性の機序は明らかになっておらず、さらなる研究が必要である。本研究で得られた成績は、今後B. gibsoniにおけるDA耐性の機序を解明する上で重要な知見と考えられる

Flow cytometry to evaluate the level of Babesia gibsoni parasitemia in vivo and in vitro by using the fluorescent nucleic acid stain SYTO16

In the present study, we employed flow cytometry to evaluate the level of parasitemia of Babesia gibsoni infecting canine erythrocytes in vivo and in vitro by using fluorescent nucleic acid staining. Peripheral blood samples from a B. gibsoni-infected dog and cultured B. gibsoni parasitizing in canine erythrocytes were stained with a membrane-permeable fluorescent nucleic acid stain, SYTO16. In this study, we utilized normal canine erythrocytes (LK erythrocytes) and canine erythrocytes containing high concentrations of potassium, reduced glutathione, and some free amino acids (HK erythrocytes) as host cells for culture. Parasitized cells in vivo were discriminated completely from unparasitized cells and a correlation (r = 0.998) between the percentage of SYTO16-positive cells and parasitemia in vivo was observed. On the other hand, erythrocytes in vitro could not be divided clearly into parasitized and unparasitized cells. However, when LK erythrocytes were used as host cells, the percentage of SYTO16-positive cells was almost the same as, and was well correlated (r = 0.932) with, the level of parasitemia. When HK erythrocytes were used as host cells, the percentage of SYTO16-positive cells was almost half of, but was correlated (r = 0.982) with, the level of parasitemia. Therefore, we attempted to observe the changes in the percentage of parasitized cells after treatment with antiprotozoal drug or mitochondria inhibitors by using flow cytometry. The changes in the percentage of SYTO16-positive cells corresponded well with the changes of the level of parasitemia when the parasites in HK erythrocytes were cultured with each compound. The present results suggest that flow cytometric detection using SYTO16 is a rapid and reliable method for monitoring parasitemia both in vivo and in vitro

Reduced transcript levels of the heat shock protein 70 gene in diminazene aceturate-resistant Babesia gibsoni variants under low concentrations of diminazene aceturate

In our previous report, we developed a diminazene aceturate (DA)-resistant Babesia gibsoni strain that was maintained in culture with 200 ng/ml DA. While developing this strain, we also obtained DA-resistant B. gibsoni variants, which were maintained in culture with DA from 1 to 175 ng/ml for more than 8 weeks. Because heat shock protein 70 (Hsp70) seems to play important roles in adaptation to a stress environment in protozoan parasites, in the present study, we examined the copy number of B. gibsoni Hsp70 (BgHsp70) transcripts of those DA-resistant variants using quantitative real-time reverse transcription-polymerase chain reaction. We found that when wild-type B. gibsoni was exposed to 1 ng/ml DA, the level of BgHsp70 transcripts was decreased at day 14. The copy number of BgHsp70 transcripts in the DA-resistant variant cultured with 1 ng/ml DA was significantly lower than in wild-type B. gibsoni, while those in DA-resistant variants increased with escalating doses of DA from 1 to 75 ng/ml, although they were lower than in wild-type B. gibsoni. However, those in DA-resistant variants cultured with ＞ 125 ng/ml DA were almost the same as wild-type B. gibsoni. These results indicated that the transcript levels of the BgHsp70 gene might be reduced when the parasites are exposed to a low concentration of DA, and then might recover to the normal level after achieving resistance against DA. We expect that further study of the function of BgHsp70 will elucidate the mechanism of drug resistance against DA in B. gibsoni

Ran-GTP assembles a specialized spindle structure for accurate chromosome segregation in medaka early embryos

Abstract Despite drastic cellular changes during cleavage, a mitotic spindle assembles in each blastomere to accurately segregate duplicated chromosomes. Mechanisms of mitotic spindle assembly have been extensively studied using small somatic cells. However, mechanisms of spindle assembly in large vertebrate embryos remain little understood. Here, we establish functional assay systems in medaka (Oryzias latipes) embryos by combining CRISPR knock-in with auxin-inducible degron technology. Live imaging reveals several unexpected features of microtubule organization and centrosome positioning that achieve rapid, accurate cleavage. Importantly, Ran-GTP assembles a dense microtubule network at the metaphase spindle center that is essential for chromosome segregation in early embryos. This unique spindle structure is remodeled into a typical short, somatic-like spindle after blastula stages, when Ran-GTP becomes dispensable for chromosome segregation. We propose that despite the presence of centrosomes, the chromosome-derived Ran-GTP pathway has essential roles in functional spindle assembly in large, rapidly dividing vertebrate early embryos, similar to acentrosomal spindle assembly in oocytes

Nutritional Supplementation Inhibits the Increase in Serum Malondialdehyde in Patients with Wet Age-Related Macular Degeneration

Purpose. To compare serum levels of malondialdehyde (MDA) in patients with wet age-related macular degeneration (wAMD), patients with dry AMD (dAMD), and patients without AMD and to evaluate the efficacy of nutritional supplementation for treating elevated serum MDA in patients with wAMD. Methods. MDA levels were measured in sera from 20 patients with wAMD, 20 with dAMD, and 24 without AMD. Patients with wAMD were randomized to receive or not receive nutritional supplementation (10 patients in each group), and MDA levels were measured after 3 months of treatment. Results. MDA levels in patients with wAMD were significantly greater compared with patients without AMD. In eyes with wAMD, there was a significant correlation between MDA levels and choroidal neovascularization lesion area. Serum MDA levels decreased in most patients that received supplementation and significantly increased in those who did not. Conclusion. Baseline serum MDA levels were elevated in patients with wAMD, and MDA levels were directly correlated with choroidal neovascularization lesion area. In addition, nutritional supplementation appeared to exert a protective effect against oxidative stress in patients with wAMD