No abstract

We previously reported that of the various polyamidoamine (PAMAM) STARBURST dendrimer (generation 3, G3) (dendrimer) conjugates with cyclodextrins (CyDs), the dendrimer (G3) conjugate with α-CyD having an average degree of substitution of 2.4 (α-CDE (G3)) has the greatest potential for a novel carrier for siRNA in vitro and in vivo. To improve the siRNA transfer activity and the lack of target specificity of α-CDE (G3), we prepared folate-polyethylene glycol (PEG)-appended α-CDEs (G3) (Fol-PαCs) with various degrees of substitution of folate (DSF) and evaluated their siRNA transfer activity to folate receptor (FR)-overexpressing cancer cells in vitro and in vivo. Of the three Fol-PαCs (G3, DSF 2, 4 and 7), Fol-PαC (G3, DSF 4) had the highest siRNA transfer activity in KB cells (FR-positive). Fol-PαC (G3, DSF 4) was endocytosed into KB cells through FR. No cytotoxicity of the siRNA complex with Fol-PαC (G3, DSF 4) was observed in KB cells (FR-positive) or A549 cells (FR-negative) up to the charge ratio of 100/1 (carrier/siRNA). In addition, the siRNA complex with Fol-PαC (G3, DSF 4) showed neither interferon response nor inflammatory response. Importantly, the siRNA complex with Fol-PαC (G3, DSF 4) tended to show the in vivo RNAi effects after intratumoral injection and intravenous injection in tumor cells-bearing mice. The FITC-labeled siRNA and TRITC-labeled Fol-PαC (G3, DSF 4) were actually accumulated in tumor tissues after intravenous injection in the mice. In conclusion, the present results suggest that Fol-PαC (G3, DSF 4) could potentially be used as a FR-overexpressing cancer cell-selective siRNA delivery carrier in vitro and in vivo.

A large number of antitumor drug delivery carriers based on passive targeting and/or active targeting have been developed. However, encapsulation of antitumor drugs into these drug carriers is often complicated, and antitumor activities of these targeting systems are not satisfactory. In the present study, we first prepared heptakis-6-folic acid (FA)-appended β-cyclodextrin (β-CyD) possessing two caproic acids between FA and a β-CyD molecule as a spacer (Fol-c(2)-β-CyD) and evaluated the potential as a novel tumor targeting carrier for antitumor drugs through a complexation. Fol-c(2)-β-CyD formed an inclusion complex with doxorubicin (DOX) at a 1:1 molar ratio with a markedly high stability constant (>10(6) M(-1)). Cellular uptake of DOX was increased by the addition of Fol-c(2)-β-CyD in KB cells, a folate receptor-α (FR-α)-positive cell line. Additionally, Fol-c(2)-β-CyD increased in vitro antitumor activities of antitumor drugs such as DOX, vinblastine (VBL), and paclitaxel (PTX) in KB cells, but not in A549 cells, a FR-α-negative cell line. The complex of DOX with Fol-c(2)-β-CyD markedly increased antitumor activity of DOX, not only after intratumoral administration but also after intravenous administration to mice subcutaneously inoculated Colon-26 cells, a FR-α-positive cell line. These findings suggest that Fol-c(2)-β-CyD could be useful as a promising antitumor drug carrier.

Tight junctions (TJs) create a paracellular permeability barrier. Although reactive oxygen species have been implicated as mediators of inflammation in inflammatory bowel diseases, their influence on the function of colonic epithelial TJs remains unknown. Oxidative stress-mediated colonic epithelial permeability was significantly attenuated by a p38 mitogen-activated protein (MAP) kinase inhibitor, SB203580. Although the amount of TJ proteins was not altered, hydrogen peroxide (H2O2) changed the localization of claudin-4 protein from an NP-40 insoluble fraction to a soluble fraction and from an apical TJ to lateral membrane. The p38 MAP kinase inactivator Wip1 significantly attenuated phosphorylation of p38 MAP kinase, and oxidative stress mediated permeability. H2O2-induced changes in claudin-4 localization were abolished by SB203580 pretreatment as well as Wip1-expressing adenovirus infection. This is the first study to demonstrate that exogenous Wip1 functions to protect oxidative stress-mediated colonic mucosal permeability and that H2O2-induced claudin-4 dislocalization is abolished by Wip1.

Aim: Work-family conflict (WFC) refers to the conflict that arises between a person's work and family life. Previous studies have reported workload, job demands, and irregular working shifts as related to the WFC among nurses and have clarified that WFC is a predictor of job satisfaction, morale, and turnover intention. Very few studies have investigated WFC among Japanese nurses and no study has taken into consideration the sense of coherence (SOC) that helps nurses to cope with stress. The present study aimed to determine the relationship between WFC and SOC and to clarify how WFC and a SOC influence the mental and physical health of nurses in order to suggest ways of establishing work environments that enable nurses to achieve a balance between their work and family life. Methods: A self-report questionnaire survey of 388 Japanese female nurses was conducted. The data from 138 nurses who were a mother and/or wife were analyzed. Results: Work-family conflict was significantly related to the SOC. It had a larger impact on the physical and mental health of nurses than their work and family characteristics. The SOC also had a major influence on the physical and mental health of nurses, while having a buffering effect on WFC with respect to depression. Conclusions: Our findings underscore the importance of taking organizational steps to create work environments that contribute to an enhanced SOC in order to reduce the WFC among nurses.

No abstract

Corn cystatin (CC), a phytocystatin, shows a wide inhibitory spectrum against various cysteine proteinases. We produced transgenic rice plants by introducing CC cDNA under CaMV 35S promoter as a first step to obtain a rice plant with insecticidal activity. This attempt was based on the observation that many insect pests, especially Coleoptera, have cysteine proteinases, probably digestive enzymes, and also that oryzacystatin, an intrinsic rice cystatin, shows a narrow inhibition spectrum and is present in ordinary rice seeds in insufficient amounts to inhibit the cysteine proteinases of rice insect pests. The transgenic rice plants generated contained high levels of CC mRNA and CC protein in both seeds and leaves, the CC protein content of the seed reaching ca. 2% of the total heat soluble protein. We also recovered CC activity from seeds and found that the CC fraction efficiently inhibited both papain and cathepsin H, whereas the corresponding fraction from non-transformed rice seeds showed much lower or undetectable inhibitory activities against these cysteine proteinases. Furthermore, CC prepared from transgenic rice plants showed potent inhibitory activity against proteinases that occur in the gut of the insect pest, Sitophilus zeamais.

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