Detergent-resistant Plasma Membrane Proteome in Oat and Rye: Similarities and Dissimilarities between Two Monocotyledonous Plants

The plasma membrane (PM) is involved in important cellular processes that determine the growth, development, differentiation, and environmental signal responses of plant cells. Some of these dynamic reactions occur in specific domains in the PM. In this study, we performed comparable nano-LC–MS/MS-based large-scale proteomic analysis of detergent-resistant membrane (DRM) fractions prepared from the PM of oat and rye. A number of proteins showed differential accumulation between the PM and DRM, and some proteins were only found in the DRM. Numerous proteins were identified as DRM proteins in oat (219 proteins) and rye (213 proteins), of which about half were identified only in the DRM. The DRM proteins were largely common to those found in dicotyledonous plants (<i>Arabidopsis</i> and tobacco), which suggests common functions associated with the DRM in plants. Combination of semiquantitative proteomic analysis and prediction of post-translational protein modification sites revealed differences in several proteins associated with the DRM in oat and rye. It is concluded that protein distribution in the DRM is unique from that in the PM, partly because of the physicochemical properties of the proteins, and the unique distribution of these proteins may define the functions of the specific domains in the PM in various physiological processes in plant cells

Detergent-resistant Plasma Membrane Proteome in Oat and Rye: Similarities and Dissimilarities between Two Monocotyledonous Plants

The plasma membrane (PM) is involved in important cellular processes that determine the growth, development, differentiation, and environmental signal responses of plant cells. Some of these dynamic reactions occur in specific domains in the PM. In this study, we performed comparable nano-LC–MS/MS-based large-scale proteomic analysis of detergent-resistant membrane (DRM) fractions prepared from the PM of oat and rye. A number of proteins showed differential accumulation between the PM and DRM, and some proteins were only found in the DRM. Numerous proteins were identified as DRM proteins in oat (219 proteins) and rye (213 proteins), of which about half were identified only in the DRM. The DRM proteins were largely common to those found in dicotyledonous plants (<i>Arabidopsis</i> and tobacco), which suggests common functions associated with the DRM in plants. Combination of semiquantitative proteomic analysis and prediction of post-translational protein modification sites revealed differences in several proteins associated with the DRM in oat and rye. It is concluded that protein distribution in the DRM is unique from that in the PM, partly because of the physicochemical properties of the proteins, and the unique distribution of these proteins may define the functions of the specific domains in the PM in various physiological processes in plant cells

Delivery of Topically Applied Calpain Inhibitory Peptide to the Posterior Segment of the Rat Eye

<div><p>We developed an inhibitory peptide that specifically acts against mitochondrial μ-calpain (Tat-μCL, 23 amino acid, 2857.37 Da) and protects photoreceptors in retinal dystrophic rats. In the present study, we topically administered Tat-μCL to the eyes of Sprague-Dawley rats for 7 days to determine both the delivery route of the peptide to the posterior segment of the eye and the kinetics after topical application in adult rats. Distribution of the peptide was determined by immunohistochemical analysis, and enzyme-linked immune-absorbent assay was used to quantify the accumulation in the retina. Peptides were prominently detected in both the anterior and posterior segments of the eye at 1 h after the final eye drop application. Immunohistochemically positive reactions were observed in the retina, optic nerve, choroid, sclera and the retrobulbar tissues, even in the posterior portion of the eye. Immunoactivities gradually diminished at 3 and 6 h after the final eye drop. Quantitative estimations of the amount of peptide in the retina were 15.3, 5.8 and 1.0 pg/μg protein at 1, 3 and 6 h after the final instillation, respectively. Current results suggest that while the topically applied Tat-μCL peptide reaches the posterior segment of the retina and the optic nerve, the sufficient concentration (> IC50) is maintained for at least 6 h in the rat retina. Our findings suggest that delivery of topically applied peptide to the posterior segment and optic nerve occurs through the conjunctiva, periocular connective tissue, sclera and optic nerve sheath.</p></div

Detergent-resistant Plasma Membrane Proteome in Oat and Rye: Similarities and Dissimilarities between Two Monocotyledonous Plants

The plasma membrane (PM) is involved in important cellular processes that determine the growth, development, differentiation, and environmental signal responses of plant cells. Some of these dynamic reactions occur in specific domains in the PM. In this study, we performed comparable nano-LC–MS/MS-based large-scale proteomic analysis of detergent-resistant membrane (DRM) fractions prepared from the PM of oat and rye. A number of proteins showed differential accumulation between the PM and DRM, and some proteins were only found in the DRM. Numerous proteins were identified as DRM proteins in oat (219 proteins) and rye (213 proteins), of which about half were identified only in the DRM. The DRM proteins were largely common to those found in dicotyledonous plants (<i>Arabidopsis</i> and tobacco), which suggests common functions associated with the DRM in plants. Combination of semiquantitative proteomic analysis and prediction of post-translational protein modification sites revealed differences in several proteins associated with the DRM in oat and rye. It is concluded that protein distribution in the DRM is unique from that in the PM, partly because of the physicochemical properties of the proteins, and the unique distribution of these proteins may define the functions of the specific domains in the PM in various physiological processes in plant cells

Distribution of topically instilled Tat-μCL in the anterior segment of the rat eye at 1 h (upper) and 3 h (lower) after the final eye drop application.

<p>Peptides are detected in the cornea, sclera, iris, ciliary body, choroid and RPE at the 1 h time point. The peptides also accumulated in the retina at the 1 h time point. The immunoreactivity weakened at the 3 h time point. DAPI: 4μ 6-diamidino-2-phenylindole. White bars indicate a 200 μm length. Results are representative of three independent experiments [n = 3 eyes (3 rats)].</p

Identification of 2-phenylethanol with a rose-like odor from anal sac secretions of the small Indian mongoose (<i>Herpestes auropunctatus</i>)

<p>The small Indian mongoose (<i>Herpestes auropunctatus</i>) is an invasive species in Okinawa and Amami-Oshima, Japan. Major strategies for their eradication have been the use of baited traps, which suffer from decreasing efficiency with declining populations and the bycatch of native animals. To address these concerns, mongoose-specific lures are required. In this study, we aimed to identify species- and/or sex-specific compounds from anal sac secretions of small Indian mongooses. Volatile compounds emitted from male and female mongoose anal sac secretions were analyzed by thermal desorption-gas chromatography-mass spectrometry. In addition to several fatty acids, 2-phenylethanol was identified as a minor compound, which is uncommon in mammalian secretions but a dominant odorant in roses. Female samples emitted higher levels of 2-phenylethanol than male samples did. These findings indicate that 2-phenylethanol is a female-specific volatile compound of anal sac secretions in small Indian mongooses, and it may be useful as an ingredient of mongoose-specific scent lures.</p> <p>2-Phenylethanol is an indicator of female anal sac secretions in small Indian mongooses.</p

Effects of an intravitreal injection or eye drop applications of Tat-µCL on ERG in S334ter rats.

<p>S334ter rats received an intravitreal injection of 2 µl of 20 mM Tat-µCL at PN 15 days (▪). Another group of S334ter rats received eye-drops containing 20 mM Tat-µCL from PN 13 to 55 days (•). Scotopic ERGs were recorded at PN 18, 21, 24, 28, 35, 42, 49, and 56 days. A) Mean amplitudes of photoreceptor-derived a-waves. B) Mean amplitudes of Müller cells-derived b-waves. Data are expressed as means ± standard deviation (n = 8 eyes (8 rats) per group). *<i>P</i><0.05 and **<i>P</i><0.01 versus the none-treated group (○) (<i>t</i>-test).</p

Effects of intravitreal injection of Tat-µCL on photoreceptor cell death in S334ter rats.

<p>A) TUNEL staining of retinal sections of S334ter rats treated with Tat-µCL. S334ter rats received intravitreal injection of 2 µl of vehicle (PBS), 4 mM PD150606, or 20 mM Tat-µCL at PN 15 days. Eyes were enucleated at PN 18 days. Retinal sections were stained with TUNEL (green) and DAPI (blue). B) Quantitative analysis of the number of TUNEL-positive cells in the ONL at PN 18 days. Data are expressed as means ± standard deviation (n = 12 eyes (6 rats) per group). ***<i>P</i><0.001 versus vehicle (<i>t</i>-test). Abbreviations: ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.</p

Effects of eye drop applications of Tat-µCL on photoreceptor cell death in P23H rats.

<p>A) TUNEL of retinal sections of P23H rats treated with eye-drops containing Tat-µCL. Eye-drops containing saline (PBS), 1 mM Tat-µCL in saline, or 1 mM Tat-µCL in 0.1% HA were administered from PN 14 to 49 days. Eyes were enucleated at PN 30, 40, or 50 days. Retinal sections were stained with TUNEL (green) and DAPI (blue). B) Quantitative analysis of the number of TUNEL-positive cells in the ONL at PN 30, 40, and 50 days. Data are expressed as means ± standard deviation (n = 12 eyes (6 rats) per group). *<i>P</i><0.05 and **<i>P</i><0.01 versus the saline-treated group (<i>t</i>-test). Abbreviations: ONL, outer nuclear layer.</p

Determination of nuclear translocation of AIF in P23H rat retinas.

<p>A) Eyes were enucleated at PN 40 days, and retinal sections were stained with AIF (red), TUNEL (green) and DAPI (blue). AIF was detected in photoreceptor cell nuclei. Arrows indicate localization of AIF in TUNEL-positive photoreceptor nuclei. B) Effects of eye-drop applications of Tat-µCL on nuclear translocation of AIF in P23H rats. Eye-drops containing saline (PBS), 1 mM Tat-µCL in saline, or 1 mM Tat-µCL in 0.1% HA were administered from PN 14 to 39 days. Eyes were enucleated at PN 40 days. Retinal sections were stained with AIF (red) and DAPI (blue). White circles indicate translocation of AIF inside photoreceptor nuclei (shown by pink color). Abbreviations: OS, photoreceptor outer segment; IS, photoreceptor inner segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.</p