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Kat o naked

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Although we previously generated transgenic torenia plants expressing green fluorescent protein from marine plankton CpYGFPin which bright fluorescence was easily visible at the whole plant level, the maximum excitation Kaat Kat o naked protein within the visible light spectrum required the use of Kat o naked coloured emission filter to eliminate exciting light. Kato naaked composition and improvisation, integrating acoustic and electric bass with language, analog and digital processed sounds. Insight into the Anime spanking video clips of the Solanaceae from the parental genomes of Petunia hybrida. Tada Makio sampler — Top Japanese session player. The film revolves around three female assassins who get close to their targets, primarily through seduction, before they kill them. Rakosy-Tican, E. Anthony Kiedis — Like every member of the Chili Peppers, Kiedis is only too happy to tear off his shirt at live shows. A cultivar of P. Categories : films Hong Kong action thriller films s action thriller films Cantonese-language films English-language films Hong Kong films Hong Kong martial arts films Media Asia films Girls with guns films Heroic bloodshed films Films about Kat o naked Films shot in the Philippines Films about assassinations. Meanwhile, Charlene is in a different temple praying for Katt's soul to rest in peace, and she tells the deity that she wishes to be with the one she truly loves, Jack.

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Composer, bass player and vocalist Kato Hideki has lived in New York since Besides playing and recording with some of the best musicians in the New York music scene, he has focused on his own work.

Kato intersperses composition and improvisation, integrating acoustic and electric bass with language, analog and digital processed sounds. The result is an interaction between prepared material and organic chance. His pieces resemble the unpredictability of life itself: alternating between intent and impulse, order and chaos, silence and sound.

He was selected for Artist-in-residence program at Harvestworks in New York. In he was a co-recipient of a grant from the Australia Council, and participated in a tour of Australia and Japan with the group Peril.

Co-founder of No Safety. Ogimi Gen percussion — Plays with salsa legend Tito Nieves. Founder of Japanese salsa band Orchesta de la Luz. Tanaka Michiaki percussion — Top Japanese session player. Played with Orchesta de la Luz. Tada Makio sampler — Top Japanese session player. Terasaki Keitaro piano — Composer.

His compositions have been performed in Europe and Canada. John Zorn piano — Composer and saxophone player. On this CD he played piano, which was his first instrument. I used a digital editing system to create the pieces; none of the musicians actually played together. Although they seem to be playing together, some of them have never even met.

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Kat o naked

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Thank you for visiting nature. You are using a browser version with limited support for CSS. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Help us improve our products. Sign up to take part. A Nature Research Journal. The application of fluorescent proteins in ornamental plants has lagged behind despite the recent development of powerful genetic tools.

Although we previously generated transgenic torenia plants expressing green fluorescent protein from marine plankton CpYGFP , in which bright fluorescence was easily visible at the whole plant level, the maximum excitation of this protein within the visible light spectrum required the use of a coloured emission filter to eliminate exciting light.

Here, to overcome this limitation, we generated transgenic petunia plants expressing eYGFPuv, a CpYGFP derivative exhibiting bright fluorescence under invisible ultraviolet UV light excitation, with a novel combination of transcriptional terminator plus translational enhancer. As expected, all transgenic plants exhibited brilliant green fluorescence easily visible to the naked eye without an emission filter. In addition, fluorescence expressed in transgenic petunia flowers was stable during long-term vegetative propagation.

Finally, we visually and quantitatively confirmed that transgenic petunia flowers resist to long-term exposure of UV without any damages such as fluorescence decay and withering. Thus, our whole-plant fluorescence imaging tool, that does not require high sensitive imaging equipment or special imaging conditions for observation, might be useful not only for basic plant research but also for ornamental purposes as a novel flower property.

Fluorescent proteins FPs have become essential tools for studying gene regulation and protein localization, and for live imaging of molecular interactions 1 , 2. On the contrary, ornamental applications of FPs in plants have lagged behind despite the recent development of powerful genetic tools.

Indeed, fluorescent proteins have been reported as a valuable tool for various plant research at the cellular level, in genetic studies as indicators of zygosity, and in the ecological monitoring of genetically modified GM plants by analysing the possibility of hybridization with relative species and introgression status 6 , 7 , 8 , 9.

Thus, appropriate optical filters are required to avoid detecting undesirable fluorescence. In addition, generation of fluorescence in plants, visible at the tissue or whole plant level, was exclusively limited to some crops and laboratory strains of Arabidopsis thaliana and tobacco plants 6 , 7 , 8 , 9 , 12 , 13 , 14 , 15 , 16 , 17 , while the generation of fluoresce in ornamental plants has remained controversial.

Although fluorescent Eustoma grandiflorum and Osteospermum ecklonis have been generated, their detailed characterization such as transgene copy number and fluorescence properties including resistance to illumination by ultraviolet UV light has not been performed We, and other colleagues, also succeeded in generating Torenia fournieri and Chrysanthemum morifolium flowers expressing CpYGFP, in which bright fluorescence was clearly visible at the whole-plant level under blue light excitation 21 , In fact, the need of emission filters is a major drawback, especially in ornamental applications of FPs.

Therefore, the present study aimed to generate ornamental plants with highly fluorescent flowers in which fluorescence is efficiently excited by solid-coloured invisible light, specifically UV light, and is clearly visualized at the whole plant level without an emission filter. These fluorescent flowers should accelerate the application of FPs to ornamental purposes.

In the present study, we designed an expression cassette in which the coding sequence of eYGFPuv was driven by the cauliflower mosaic virus CaMV 35S promoter combined with the transcriptional terminator of the heat shock protein We introduced a tandemly triplicated expression cassette into commercial strains of garden petunia, Petunia hybrida by Agrobacterium tumefaciens -mediated transformation and generated fluorescent transgenic Tg lines expressing eYGFPuv.

As expected, Tg plants exhibited very bright green fluorescence easily visible to the naked eye under UV excitation, and this fluorescence was kept stable by vegetative propagation.

In addition, we visually and quantitatively confirmed that transformed P. The present study aimed to generate flowers in which fluorescence is efficiently excited by solid coloured light i. Therefore, we designed a tandemly triplicated expression cassette using the approach of Sasaki et al.

Normalized Ex solid line and Em dotted line spectra are shown with Ex and Em maxima, respectively. All experiments mentioned above were performed once. Transformed juvenile plants regenerated from these calli kept brilliant florescence at the whole plant level, including stem and root fluorescence Fig. Next, we examined the copy numbers of these transgenes by southern blot analysis Fig. Both Eco RI and Xba I cut behind the probe sequence and distinguishes the head-tail tandem insertion.

We compared the fluorescence of adult P. As expected, when excited by UV light, eYGFPuv-expressing plants, especially P, exhibited strong florescence at the whole plant level, including petals, leaves, and stems, and this fluorescence was much stronger than that observed when the same plants were excited by blue visible light. On the other hand, P only exhibited bright and specific fluorescence when excited by blue visible light, which was observed using a yellowish emission filter.

The fluorescence of harvested petals and leaves is shown in Fig. Under UV excitation, only eYGFPuv-expressing petals and leaves, especially those derived from P, exhibited very bright green fluorescence, while under blue light excitation, only eYGFP-expressing P exhibited bright fluorescence. Notably, the fluorescence expressed in P petals was much stronger than that expressed in leaves.

Crude protein extracts from petals and leaves of WT and eYGFPuv-expressing transgenic lines analysed by native-PAGE revealed strong fluorescence signals of the corresponding protein band, which were in agreement with the patterns observed in visual inspection under UV light excitation Fig.

Fluorescence in transgenic Petunia hybrida plants from several lineages. Each image was taken under UV or blue light excitation as described above. In all experiments, the fluorescence excited by blue visible light was observed through a yellowish emission filter SC, Fujifilm, Tokyo, Japan.

A representative of two independent experiments using different pots is shown. Native polyacrylamide gel electrophoresis of crude protein extracts from transgenic Petunia hybrida flowers. Because of the self-incompatible nature of the F1 hybrid cultivar, we had to maintain Tg lines by vegetative propagation. However, the bright fluorescence of Tg flowers was kept for at least 18 months, i. A cultivar of P.

As expected, under UV excitation, eYGFPuv-expressing plants exhibited very bright green fluorescence with high-contrast at the whole plant level, which was easily visualized with naked eye Fig. Using our novel eYGFPuv gene with a potent combination of transcriptional terminator plus translational enhancer, we could generate green fluorescent flowers in which bright fluorescence can be easily visualized at the whole plant level without any high sensitive imaging equipment and emission filter.

Each image was taken under UV excitation as described above. Because all Tg plants tested by visual inspection presented intense fluorescence levels, transgene expression in P. According to the recent validation of reference genes in P. Although transcription levels of FP genes were 2- to 4-fold higher in leaves than in petals, the overall transcription levels of FP genes were similar among Tg P.

Regarding plant cells, FPs are now generally accepted not to be cytotoxic 6 , 8 , In addition, no growth inhibition or morphological defect was observed in the fluorescent plants generated in this study data not shown. However, because resistance to long UV exposure will be important for the application of FPs in ornamental flowers, we examined the expression level of the fluorescence gene and anti-oxidative stress responses in Tg P. Notably, all plants, including WT plants, exhibited no specific growth inhibition and withering under UV exposure during the two weeks period Fig.

Overall, these data strongly support that fluorescent P. Using our novel fluorescent genes, we have succeeded in generating P. Thus, the fluorescence of these transgenic plants was comparable to or brighter than that of other FP-expressing plants reported as visible at the tissue or whole plant level 8 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , although the detailed acquisition conditions of fluorescence images in such reports was not fully available for comparisons, nor did we directly compare the fluorescence of other GFP variants by generating Tg plants.

Interestingly, visual inspections of P. Because the red fluorescence signals of chlorophyll or chlorophyll-protein complexes observed in each leaf were considerably stronger than the corresponding green fluorescence of FPs in both native-PAGE analysis Fig.

Bright and stable fluorescence was achieved in the present study in two commercial cultivars of P. Because plants generally have weak acid intracellular environment, ranging from approximately pH 4.

However, at least in our experimental settings, which were sufficient to observe the bright fluorescence of Tg plants with the naked eye, fluorescent P. Anyway, these data greatly support the application of our fluorescence genes for the development of novel ornamental plants with bright fluorescence. We are currently developing fluorescent flowers in other horticultural plants, e. Finally, in addition to contributing to plant research, fluorescent plants have high potential for cultural and educational purposes by showing an amusing side of science.

Indeed, green fluorescent P. Therefore, these fluorescent flowers might also be a good tool to gain public acceptance of GM plants 7 , 8. Unless otherwise mentioned, all enzymes were purchased form Takara Bio Inc. Shiga, Japan. We selected P.

White-flowered P. Seeds of P. The binary vectors were introduced into A. After transformation of P. After regeneration of plants from Tg shoots, they were transferred to soil and grown in a glasshouse. A yellowish sharp cut filter SC, Fujifilm, Tokyo, Japan was used as an emission filter for blue light excitation. Wild type and P. Stepanenko, O. Fluorescent proteins as biomarkers and biosensors: throwing color lights on molecular and cellular processes. Curr Protein Pept Sci.

Shaner, N. A guide to choosing fluorescent proteins. Nat Methods. Gong, Z. Development of transgenic fish for ornamental and bioreactor by strong expression of fluorescent proteins in the skeletal muscle.

Biochem Biophys Res Commun. Knight, J. GloFish casts light on murky policing of transgenic animals. Iizuka, T. Colored fluorescent silk made by transgenic silkworms. Adv Funct Mater. Stewart, C. The utility of green fluorescent protein in transgenic plants. Plant Cell Rep.

Kat o naked

Kat o naked