Potential Use of Folate-polyethylene glycol (PEG)-Appended Dendrimer (G3) Conjugate with alpha-Cyclodextrin as DNA Carriers to Tumor Cells

We previously reported that polyamidoamine STARBURST dendrimer (generation 3, G3) (dendrimer) conjugate with alpha-cyclodextrin (alpha-CyD) having an average degree of substitution of 2.4 of alpha-CyD (alpha-CDE) provided remarkable aspects as novel carriers for DNA and siRNA. To develop novel alpha-CDE derivatives with tumor cell specificity, we prepared folate-appended alpha-CDEs (Fol-alpha-CDEs) and folate-polyethylene glycol (PEG)-appended alpha-CDEs (Fol-PalphaCs) with the various degrees of substitution of folate (DSF), and evaluated in vitro and in vivo gene transfer activity, cytotoxicity, cellular association and physicochemical properties. In vitro gene transfer activity of Fol-alpha-CDEs (G3, DSF 2, 5 or 7) was lower than that of α-CDE (G3) in KB cells, folate receptor (FR)-overexpressing cancer cells. Of the three Fol-PalphaCs (G3, DSF 2, 5 or 7), Fol-PalphaC (G3, DSF 5) had the highest gene transfer activity in KB cells. The activity of Fol-PalphaC (G3, DSF 5) was significantly higher than that of alpha-CDE (G3) in KB cells, but not in A549 cells, FR-negative cells. Negligible cytotoxicity of the pDNA complex with Fol-PalphaC (G3, DSF 5) was observed in KB cells or A549 cells up to a charge ratio of 100/1 (carrier/pDNA). The cellular association of the pDNA complex with Fol-PalphaC (G3, DSF 5) could be mediated by FR on KB cells, resulting in its efficient cellular uptake. Fol-PalphaC (G3, DSF 5) had higher binding affinity with folate binding protein (FBP) than alpha-CDE (G3), although the physicochemical properties of pDNA complex with Fol-PalphaC (G3, DSF 5) were almost comparable to that with alpha-CDE (G3), although the onset charge ratio and the compaction ability of Fol-PalphaC (G3, DSF 5) were slightly different. Fol-PalphaC (G3, DSF 5) tended to show higher gene transfer activity than alpha-CDE (G3) 12 h after intratumoral administration in mice. These results suggest that Fol-PalphaC (G3, DSF 5), not Fol-alpha-CDEs, could be potentially used as a FR-overexpressing cancer cell-selective DNA carrier

Essential role of eIF5-mimic protein in animal development is linked to control of ATF4 expression

Translational control of transcription factor ATF4 through paired upstream ORFs (uORFs) plays an important role in eukaryotic gene regulation. While it is typically induced by phosphorylation of eIF2α, ATF4 translation can be also induced by expression of a translational inhibitor protein, eIF5-mimic protein 1 (5MP1, also known as BZW2) in mammals. Here we show that the 5MP gene is maintained in eukaryotes under strong purifying selection, but is uniquely missing in two major phyla, nematoda and ascomycota. The common function of 5MP from protozoa, plants, fungi and insects is to control translation by inhibiting eIF2. The affinity of human 5MP1 to eIF2β was measured as being equivalent to the published value of human eIF5 to eIF2β, in agreement with effective competition of 5MP with eIF5 for the main substrate, eIF2. In the red flour beetle, Tribolium castaneum, RNA interference studies indicate that 5MP facilitates expression of GADD34, a downstream target of ATF4. Furthermore, both 5MP and ATF4 are essential for larval development. Finally, 5MP and the paired uORFs allowing ATF4 control are conserved in the entire metazoa except nematoda. Based on these findings, we discuss the phylogenetic and functional linkage between ATF4 regulation and 5MP expression in this group of eukaryotes

Modeling Retinal Degeneration Using Patient-Specific Induced Pluripotent Stem Cells

Retinitis pigmentosa (RP) is the most common inherited human eye disease resulting in night blindness and visual defects. It is well known that the disease is caused by rod photoreceptor degeneration; however, it remains incurable, due to the unavailability of disease-specific human photoreceptor cells for use in mechanistic studies and drug screening. We obtained fibroblast cells from five RP patients with distinct mutations in the RP1, RP9, PRPH2 or RHO gene, and generated patient-specific induced pluripotent stem (iPS) cells by ectopic expression of four key reprogramming factors. We differentiated the iPS cells into rod photoreceptor cells, which had been lost in the patients, and found that they exhibited suitable immunocytochemical features and electrophysiological properties. Interestingly, the number of the patient-derived rod cells with distinct mutations decreased in vitro; cells derived from patients with a specific mutation expressed markers for oxidation or endoplasmic reticulum stress, and exhibited different responses to vitamin E than had been observed in clinical trials. Overall, patient-derived rod cells recapitulated the disease phenotype and expressed markers of cellular stresses. Our results demonstrate that the use of patient-derived iPS cells will help to elucidate the pathogenic mechanisms caused by genetic mutations in RP

キョジュウ ケイタイ カラ ミタ ジョシ ガクセイ ノ ショクイシキ ト ショクコウドウ

居住形態別に食意識・食行動および食物摂取状況を把握するために,栄養士養成課程3年次学生を対象に,食意識と食行動に関する調査と料理単位法による食物摂取状況調査を実施した。居住形態により食事の調理担当者に違いがみられ,「家族と同居」群の場合は自分以外の家族が担当し,昼食も手作り弁当が多くなっていた。昼食に購入品が多い「一人暮らし」群に比べ,昼食においてほとんどの栄養素等摂取量が有意な高値を示し,また,1日当たりの摂取量も有意な高値を示した。1日当たり食品群別摂取量においても「家族と同居」群の方が,米類,いも類,緑黄色野菜類,その他野菜類,きのこ類,海藻類の摂取量が有意に多くなっていた。食事バランスガイドのサービング数でみると,「家族と同居」群でも摂りたい目安のサービング数に比べ主菜以外の摂取が少なく,「一人暮らし」群は,さらに少なくなっていた。「一人暮らし」群の中で食事の栄養バランスが取れていると思う者は15.4%で,「家族と同居」群に比べて低く,有意差が認められた。居住形態にかかわらず食事を改善したいという意欲はあるが,時間やお金の制約により改善できないということが明らかになった。したがって,望ましい食物摂取のためには内食に限らず,中食,外食を含めて,個々が実践できるよう食スキルの育成と食環境を整えていく必要があると考える。The purpose of this study is to determine the current state of dietary consciousness and habits and food intake in relation to living style. A survey on these three factors was administered to 165 college students enrolled in a nutrition course in Tokyo. Compared with students living alone, students living at home often brought their own lunches although they cooked less often. In addition, the intake of nutrients by students living at home was significantly higher. Students living with their family were also found to consume significantly more quantities of rice, potatoes, vegetables, mushrooms, and seaweed. In general, the students were found to consume less than the number of servings for each food group (except for main dishes) recommended in the Japanese Food Guide Spinning Top, with students living alone consuming even less. Students who were in control of their dietary habits made up 15.4% of those living alone, and this percentage was significantly lower than of those living at home. Most of the students wanted to follow ideal dietary habits, but could not afford the money and time to do so. The results suggest that the cultivation of an environment that matches participants\u27 characteristics could improve their dietary habits

Seismic Exploration Using Active Sources at Kuchierabujima Volcano, Southwest Japan

Seismic exploration using artificial sources was conducted at Kuchierabujima volcano, southwest Japan in November 2004 by 40 participants from 9 national universities andJapan Meteorological Agency to investigate the subsurface seismic structure. The exploration was the 11th joint experiment under the National Project for Prediction of Volcanic Eruptions. A total of 183 temporal stations equippedwith a 2 Hz vertical component seismometer (including 75 3component seismometers) and a portable data logger were deployed on Kuchierabu Island. Dynamite shots with charges of 10-115 kg were detonated at 19 locations, and seismic signals were successfully recorded. To reveal the P-wave velocity structure, 2955 arrival times of the first motion were picked from the seismograms, and 2187 were classified into ranks A and B. From the record sections and the arrival time data, characteristics reflecting the geological structure were identified. Refracted waves of 5 km/s were observed at stations＞5km from the shot points. Apparent velocities near the shot points depend on the surface geology around the shots. P-wave arrived earlier at stations near the summits. Strongly scattered waves were observed similarly near the summits

Stepwise differentiation of pluripotent stem cells into retinal cells

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage embryos. They can maintain an undifferentiated state indefinitely and can differentiate into derivatives of all three germ layers, namely ectoderm, endoderm and mesoderm. Although much progress has been made in the propagation and differentiation of ES cells, induction of photoreceptors has generally required coculture with or transplantation into developing retinal tissue. Here, we describe a protocol for generating retinal cells from ES cells by stepwise treatment with defined factors. This method preferentially induces photoreceptor and retinal pigment epithelium (RPE) cells from mouse and human ES cells. In our protocol, differentiation of RPE and photoreceptors from mouse ES cells requires 28 d and the differentiation of human ES cells into mature RPE and photoreceptors requires 120 and 150 d, respectively. This differentiation system and the resulting pluripotent stem cell-derived retinal cells will facilitate the development of transplantation therapies for retinal diseases, drug testing and in vitro disease modeling. It will also improve our understanding of the development of the central nervous system, especially the eye