Gene expression of PLAT and ATS3 proteins increases plant resistance to insects.

MAIN CONCLUSION: Genes of the PLAT protein family, including PLAT and ATS3 subfamilies of higher plants and homologs of liverwort, are involved in plant defense against insects. Laticifer cells in plants contain large amounts of anti-microbe or anti-insect proteins and are involved in plant defense against biotic stresses. We previously found that PLAT proteins accumulate in laticifers of fig tree (Ficus carica) at comparable levels to those of chitinases, and the transcript level of ATS3, another PLAT domain-containing protein, is highest in the transcriptome of laticifers of Euphorbia tirucalli. In this study, we investigated whether the PLAT domain-containing proteins are involved in defense against insects. Larvae of the lepidopteran Spodoptera litura showed retarded growth when fed with Nicotiana benthamiana leaves expressing F. carica PLAT or E. tirucalli ATS3 genes, introduced by agroinfiltration using expression vector pBYR2HS. Transcriptome analysis of these leaves indicated that ethylene and jasmonate signaling were activated, leading to increased expression of genes for PR-1, ß-1,3-glucanase, PR5 and trypsin inhibitors, suggesting an indirect mechanism of PLAT- and ATS3-induced resistance in the host plant. Direct cytotoxicity of PLAT and ATS3 to insects was also possible because heterologous expression of the corresponding genes in Drosophila melanogaster caused apoptosis-mediated cell death in this insect. Larval growth retardation of S. litura occurred when they were fed radish sprouts, a good host for agroinfiltration, expressing any of nine homologous genes of dicotyledon Arabidopsis thaliana, monocotyledon Brachypodium distachyon, conifer Picea sitchensis and liverwort Marchantia polymorpha. Of these nine genes, the heterologous expression of A. thaliana AT5G62200 and AT5G62210 caused significant increases in larval death. These results indicated that the PLAT protein family has largely conserved anti-insect activity in the plant kingdom (249 words).

Effect of topically applied sphingomyelin-based liposomes on the ceramide level in a three-dimensional cultured human skin model.

Sphingomyelin-based liposomes were prepared and applied to the stratum corneum side or basal layer side of a three-dimensional (3D) cultured human skin model, and the increase in the type II ceramide (ceramide II) content of the cultured skin model was evaluated. The sphingomyelin-based liposomes were prepared by a high-pressure emulsification method, and the obtained liposomes were characterized; the particle diameter and zeta potential of the liposomes were 155.3 nm and -11.4 mV, respectively. Their spherical shape and lamella structure were observed by transmission electron microscopy. The sphingomyelin-based liposomes or saline were applied to the cultured skin model, and ceramide II was extracted from the skin model. The extracted ceramide II was separated by high-performance thin-layer chromatography and quantified by a densitometer. The amount of ceramide II in the cultured skin model was significantly increased by the application of the sphingomyelin-based liposomes, compared with the nonapplication group. Thus, sphingomyelin-based liposomes are useful for enriching the ceramide level in 3D cultured skin models.

Radish sprouts as an efficient and rapidly available host for an agroinfiltration-based transient gene expression system.

Agroinfiltration, the infiltration of plants with Agrobacterium harboring a plasmid that contains a specific gene, is used to transiently express a gene in a heterologous organism. Using the "Tsukuba system", greater amounts of target protein accumulate compared with usual expression plasmids. Reported host plants, including Nicotiana benthamiana, a common plant for agroinfiltration, need several weeks after sowing to grow enough for infection. To shorten the culture period and, thereby, improve target protein production, we tested sprouts as host plants. Sprouts were grown in the dark to encourage elongation so that vacuum infiltration becomes easier, and this was followed by a few days of exposure to illumination before infection with pBYR2HS-EGFP, the EGFP expression plasmid of the Tsukuba system. Among six tested species of Fabaceae and Brassicaceae, radish showed the highest transient expression. Among six tested radish cultivars, Kaiware, Hakata, and Banryoku provided the best results. Culturing for 5 day, including 1 day of imbibition and 1 to 2 day of exposure to illumination resulted in EGFP fluorescence in 80% of the cotyledon area. Thus, a remarkable amount of EGFP was obtained only 8 day after seed imbibition. The EGFP amount in Kaiware cotyledons was comparable with Rubisco at ∼0.7 mg/g fresh weight. Kaiware sold in supermarkets could also be used, but resulted in lower expression levels.

[Low back load reduction using mechanical lift during transfer of patients].

In Japanese care-work sites, care-workers (CWs) have lacked basic health risk awareness for transferring patients. Knowledge of lifting equipment and skills for transfer of patients have not been disseminated and many CWs have suffered from work-related musculoskeletal disorders, especially low back pain (LBP). In order to find better ways of patient transfer which reduce and prevent LBP, we conducted a study of low back loads and operation time during the transfer of a simulated patient, who was totally dependent from bed to wheelchair, using a mechanical lift (Lift) and manual handling (handling). Moreover we examined the levels of skill which CWs had acquired in transfer by Lift and the effects of acquired skill on low back loads and operation time. We explored low back load using surface electromyography (EMG) of the lumbar paraspinals between L3 and L4 and the trunk inclination angle (TIA) measurement method. The subjects were 5 caregivers who performed the task of transferring a simulated patient from lying on the bed to sitting in a wheelchair using the Lift and by handling. Handling transfer was assisted by two-persons at the head and foot. A 'simulated' patient (a 70 kg healthy male; instructed to keep whole body relaxed) was used in all transfer tasks. When subjects used the Lift, we made an ergonomics checklist for reduction of low back load of caregivers. Subjects performed the task 4 times and were evaluated with the checklist. The level of acquired skill was significantly improved by the guidance of the checklist. TIA was observed to be significantly lower in Lift than in handling, but with EMG no significant differences were seen between Lift and handling. The effects of acquired skill on low back loads showed that TIA was statistically reduced at high skill as compared to low skill. However, there were no significant differences between both skills in Lift and handling by EMG. Operation time of Lift showed significant shortening of operation time with high skill as compared to low skill. Operation time of Lift was about 10 times longer than handling. Thus, we suggest that transfer by Lift is a valid way of reducing the burden on CWs low back. Additionally, this study found that for reduction of LBP risk for CWs, it will be important not only to use the Lift but also to observe proper procedure and raise CW skill levels in patient transfer.

Tumor targeting of doxorubicin by anti-MT1-MMP antibody-modified PEG liposomes.

Immunoliposomes are potent carriers for targeting of therapeutic drugs to specific cells. Membrane type-1 matrix metalloproteinase (MT1-MMP), which plays an important role in angiogenesis, is expressed on angiogenic endothelium cells as well as tumor cells. Then, the MT1-MMP might be useful as a target molecule for tumor and neovascularity. In the present study, we addressed a utility of antibodies against the MT1-MMP as a targeting ligand of liposomal anticancer drug. Fab' fragments of antibody against the MT1-MMP were modified at distal end of polyethylene glycol (PEG) of doxorubicin (DXR)-encapsulating liposomes, DXR-sterically stabilized immunoliposomes (DXR-SIL[anti-MT1-MMP(Fab')]). Modification with the antibody significantly enhanced cellular uptake of DXR-SIL[anti-MT1-MMP(Fab')] into the HT1080 cells, which highly express MT1-MMP, compared with the non-targeted liposomes (DXR-stealthliposomes (DXR-SL)), suggesting that MT1-MMP antibody (Fab') is a potent targeting ligand for the MT1-MMP expressed cells. In vivo systemic administration of DXR-SIL[anti-MT1-MMP(Fab')] into the tumor-bearing mice showed significant suppression of tumor growth compared to DXR-SL. This is presumably due to the active targeting of immunoliposomes for tumor and neovascularity. However, tumor accumulation of DXR-SIL[anti-MT1-MMP(Fab')] and DXR-SL were comparable, suggesting that both liposomal formulations accumulated in tumor via enhanced permeation and retention (EPR) effect, but not via targeting to the MT1-MMP expressed on both the endothelial and tumor cells. It appears that the enhanced antitumor activity of DXR-SIL[anti-MT1-MMP(Fab')] resulted from acceleration of cellular uptake of lioposomes owing to the incorporated antibody after extravasation from capillaries in tumor.

Highly water-soluble matrix metalloproteinases inhibitors and their effects in a rat adjuvant-induced arthritis model.

A new series of succinate-based dual inhibitors against matrix metalloproteinases (MMPs) and tumor necrosis factor alpha converting enzyme (TACE) possessing highly-water solubility was designed, synthesized, and evaluated for enzyme inhibition. Incorporating of acidic or basic functional groups at the P(2)' position afforded sufficient water solubility without significant loss of inhibitory potencies. Compound 18e, which had a guanidino group at the P(2)' position as the basic functional group, exhibited broad inhibition against target enzymes for a relatively long period in rat plasma (beta t(1/2); 2.0h) after sc administration when compared with compounds possessing acidic functional groups (18a and 18b). Consequently, the representative compound 18e together with compound 18b, Marimastat and Trocade were evaluated in the rat adjuvant-induced arthritis model, a model of chronic cartilage destruction. It is concluded that the newly synthesized highly water-soluble compound 18e showed significant activity in suppressing hindpaw swelling and the bone destruction with a minimal administration period (days 3-7).

Design and synthesis of sulfur based inhibitors of matrix metalloproteinase-1.

Fibroblast collagenase (MMP-1), a member of the matrix metalloproteinases family, is believed to be a pathogenesis of arthritis, by cleaving triple-helical type II collagen in cartilage. From the similarity of the active site zinc binding mode with hydroxamate, we designed and synthesized alpha-mercaptocarbonyl possessing compounds (3-5), which incorporated various peptide sequences as enzyme recognition sites. The P4-P1 peptide incorporating compound (3) exhibited as potent inhibition as the hydroxamate (1) and the carboxylate (2) type inhibitors, with an IC50 of 10(-6) M order against MMP-1. But the inhibitor (3) related compounds (6-8) displayed decreased or no inhibitory potencies. These results suggest that the existence of both the carbonyl and thiol groups might be critical for the inhibition, and the distance between the two functional groups is important for inhibitory potency. For Pn' peptide incorporating compounds (4a-k), except for 4h and 4k, all compounds showed IC50 values under sub-nanomolar. Among them, for potent inhibition, Leu was better than Phe and Val as the P1' amino acid, and the P2' position amino acid was necessary, and preferentially Phe. Insertion of the Pn peptide into 4d or 4k, giving compounds 5a-c, did not increase the activities of 4d and 4k. Substitution of the mercapto group with other functional groups lost the activity of compound 4a. The stereochemical preference at the thiol-attached position was also determined by preparation of both isomers of 4a. It was found that the S configuration compound (36b) is approximately 100 times more potent than the corresponding R-isomer (36a).

Suppressor mechanism of serum thymic factor on tumor necrosis factor-alpha-induced apoptosis in the mouse pancreatic beta-cell line.

Tumor necrosis factor-alpha (TNF-alpha) is a cytokine considered to play a key role in beta-cell destruction in insulin-dependent diabetes mellitus (IDDM). Serum thymic factor (Facteur thymique serique; FTS) is a nonapeptide thymus hormone known to inhibit IDDM in a mouse model. In this study, the effect of TNF-alpha on the murine pancreatic beta-cell line MIN6 was examined. Cell shrinkage and detachment were seen in cells treated with 0-50 ng/ml TNF-alpha for 12h. Oligonucleosomal DNA fragmentation was determined from non-adherent cells, indicating that the TNF-alpha-induced cell destruction was attributed to apoptosis. Fragmented DNA was quantified by enzyme-linked immunosorbent assay to measure the amount of histone-bound oligonucleosomes. FTS was treated with TNF-alpha and the percentage of fragmented DNA was analyzed. The data indicate a distinct reduction of fragmented DNA at a concentration of 1 ng/ml FTS. Expression of TNF receptor I, inducible form of nitric oxide synthase (iNOS), interleukin-1 beta-converting enzyme (ICE), Bcl-2, and nuclear factor kappa B (NF-kappa B) was analyzed by reverse transcriptase-polymerase chain reaction to investigate the suppressor mechanism of FTS on TNF-alpha-induced apoptosis. FTS treatment suppressed the expression of iNOS and Bcl-2 mRNA in TNF-alpha-treated cells. The expression of NF-kappa B mRNA in TNF-alpha-treated cells was enhanced after FTS treatment, while that of ICE mRNA did not change in TNF-alpha-treated cells with or without FTS treatment. These results suggest that the inhibition of MIN6 cell death by FTS on TNF-alpha-induced apoptosis is caused by a negative feedback mechanism involving the inhibition of iNOS induction.