ROCK Inhibitor (Y-27632) Abolishes the Negative Impacts of miR-155 in the Endometrium-Derived Extracellular Vesicles and Supports Embryo Attachment.

Extracellular vesicles (EVs) are nanosized vesicles that act as snapshots of cellular components and mediate cellular communications, but they may contain cargo contents with undesired effects. We developed a model to improve the effects of endometrium-derived EVs (Endo-EVs) on the porcine embryo attachment in feeder-free culture conditions. Endo-EVs cargo contents were analyzed using conventional and real-time PCR for micro-RNAs, messenger RNAs, and proteomics. Porcine embryos were generated by parthenogenetic electric activation in feeder-free culture conditions supplemented with or without Endo-EVs. The cellular uptake of Endo-EVs was confirmed using the lipophilic dye PKH26. Endo-EVs cargo contained miR-100, miR-132, and miR-155, together with the mRNAs of porcine endogenous retrovirus (PERV) and ß-catenin. Targeting PERV with CRISPR/Cas9 resulted in reduced expression of PERV mRNA transcripts and increased miR-155 in the Endo-EVs, and supplementing these in embryos reduced embryo attachment. Supplementing the medium containing Endo-EVs with miR-155 inhibitor significantly improved the embryo attachment with a few outgrowths, while supplementing with Rho-kinase inhibitor (RI, Y-27632) dramatically improved both embryo attachment and outgrowths. Moreover, the expression of miR-100, miR-132, and the mRNA transcripts of BCL2, zinc finger E-box-binding homeobox 1, ß-catenin, interferon-γ, protein tyrosine phosphatase non-receptor type 1, PERV, and cyclin-dependent kinase 2 were all increased in embryos supplemented with Endo-EVs + RI compared to those in the control group. Endo-EVs + RI reduced apoptosis and increased the expression of OCT4 and CDX2 and the cell number of embryonic outgrowths. We examined the individual and combined effects of RI compared to those of the miR-155 mimic and found that RI can alleviate the negative effects of the miR-155 mimic on embryo attachment and outgrowths. EVs can improve embryo attachment and the unwanted effects of the de trop cargo contents (miR-155) can be alleviated through anti-apoptotic molecules such as the ROCK inhibitor.

Dynamic proteomic profile of potato tuber during its in vitro development.

Potato tuberization is a complicated biochemical process, which is dependent on external environmental factors. Tuber development in potato consists of a series of biochemical and morphological processes at the stolon tip. Signal transduction proteins are involved in the source-sink transition during potato tuberization. In the present study, we examined protein profiles under in vitro tuber-inducing conditions using a shotgun proteomic approach involving denaturing gel electrophoresis and liquid chromatography-mass spectrometry. A total of 251 proteins were identified and classified into 9 groups according to distinctive expression patterns during the tuberization stage. Stolon stage-specific proteins were primarily involved in the photosynthetic machinery. Proteins specific to the initial tuber stage included patatin. Proteins specific to the developing tuber stage included 6-fructokinase, phytoalexin-deficient 4-1, metallothionein II-like protein, and malate dehydrogenase. Novel stage-specific proteins identified during in vitro tuberization were ferredoxin-NADP reductase, 34 kDa porin, aquaporin, calmodulin, ripening-regulated protein, and starch synthase. Superoxide dismutase, dehydroascorbate reductase, and catalase I were most abundantly expressed in the stolon; however, the enzyme activities of these proteins were most activated at the initial tuber. The present shotgun proteomic study provides insights into the proteins that show altered expression during in vitro potato tuberization.

Proteomic analysis of Korean ginseng (Panax ginseng C.A. Meyer).

Although many reports have been published regarding the pharmacological effects of ginseng, little is known about the biochemical pathways operant in ginsenoside biosynthesis, or the genes involved therein. Proteomics analysis is an approach to elucidate the physiological characteristics and biosynthetic pathways of ginsenosides, main components of ginseng. In this review, we introduced the recent progress in proteomics studies of ginseng (Panax ginseng C.A. Meyer). We briefly reference the genomic analyses of P. ginseng, without which proteomics approaches would have been impossible. Functional genomics studies regarding secondary metabolism in P. ginseng are also introduced here, in order to introduce possible future prospects for further study.

Characterization of RNase-like major storage protein from the ginseng root by proteomic approach.

The most abundant root proteins of ginseng (Panax ginseng) have been detected and identified by comparative proteome analysis with cultured hairy root of ginseng. Four abundant proteins (28, 26, 21 and 20 kDa) of P. ginseng had isoforms with different pl values on two-dimensional gel electrophoresis (2DE). The results of N-terminal and internal amino acid sequencing, however, showed that all of them originate from a 28 kDa protein, known as ginseng major protein (GMP). The GMP gene was searched for in the expressed sequence tag database of P. ginseng and found to encode a 27.3 kDa protein having 238 amino acid residues. Analysis of the amino acid sequences indicates that GMP exhibits high sequence homology with plant RNases and RNase-like proteins. However, purified GMP had no RNase activity even though it has conserved amino acid residues known to be essential for active sites of RNase. The GMPs present in ginseng main root were not expressed in cultured hairy roots of ginseng. 2DE analysis showed that the amounts of GMPs in main roots change according to seasonal fluctuation. These results suggest that the GMPs are root-specific RNase-like proteins, which function as vegetative storage proteins of ginseng for survival in the natural environment.

Proteome analysis of the responses of Panax ginseng C. A. Meyer leaves to high light: use of electrospray ionization quadrupole-time of flight mass spectrometry and expressed sequence tag data.

We performed comparative proteomic analyses in order to understand the physiological responses of ginseng (Panax ginseng C. A. Meyer) to high light (HL). As a first step, we analyzed the proteins expressed in ginseng leaves. Proteins extracted from leaves were separated by two-dimensional polyacrylamide gel electrophoresis. Protein spots were identified by tandem mass spectra analysis using electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS). We used a ginseng expressed sequence tag (EST) database as well as a nonredundant protein database from NCBI to identify proteins. Eighty-one proteins were identified using the nr protein database, 51 of which were also verified from the ginseng EST database. An additional 66 proteins were identified only from the ginseng EST database. Proteins that function in energy metabolism, protein stabilization, and protection against oxidative stress were abundant. To understand the light responses of ginseng leaves, we studied time dependent changes in expressed proteins produced by 0-4 h of HL exposure. Six HL-responsive proteins were identified: three proteins were up-regulated (cytosolic small heat-shock protein, cytosolic ascorbate peroxidase, and putative major latex-like protein) and three proteins were down-regulated (Rieske Fe/S protein, putative 3-beta hydroxysteroid dehydrogenase/isomerase-like protein, and oxygen-evolving enhancer-like protein). Our results show that the ginseng EST database combined with ESI Q-TOF MS analysis can be used to identify ginseng proteins and to elucidate the protective mechanism of ginseng against HL induced damage.

Proteome analysis of hairy root from Panax ginseng C.A. Meyer using peptide fingerprinting, internal sequencing and expressed sequence tag data.

As an initial step to the comprehensive proteomic analysis of Panax ginseng C. A. Meyer, protein mixtures extracted from the cultured hairy root of Panax ginseng were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). The protein spots were analyzed and identified by peptide finger printing and internal amino acid sequencing by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS), respectively. More than 300 protein spots were detected on silver stained two-dimensional (2-D) gels using pH 3-10, 4-7, and 4.5-5.5 gradients. Major protein spots (159) were analyzed by peptide fingerprinting or de novo sequencing and the functions of 91 of these proteins were identified. Protein identification was achieved using the expressed sequence tag (EST) database from Panax ginseng and the protein database of plants like Arabidopsis thaliana and Oryza sativa. However, peptide mass fingerprinting by MALDI-TOF MS alone was insufficient for protein identification because of the lack of a genome database for Panax ginseng. Only 17 of the 159 protein spots were verified by peptide mass fingerprinting using MALDI-TOF MS whereas 87 out of 102 protein spots, which included 13 of the 17 proteins identified by MALDI-TOF MS, were identified by internal amino acid sequencing using tandem mass spectrometry analysis by ESI Q-TOF MS. When the internal amino acid sequences were used as identification markers, the identification rate exceeded 85.3%, suggesting that a combination of internal sequencing and EST data analysis was an efficient identification method for proteome analysis of plants having incomplete genome data like ginseng. The 2-D patterns of the main root and leaves of Panax ginseng differed from that of the cultured hairy root, suggesting that some proteins are exclusively expressed by different tissues for specific cellular functions. Proteome analysis will undoubtedly be helpful for understanding the physiology of Panax ginseng.