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11.
cDNA cloning of an extracellular dermal glycoprotein of carrot and its expression in response to wounding 总被引:2,自引:0,他引:2
Suspension-cultured cells of carrot (Daucus carota L.) synthesize and secrete a glycoprotein that is normally found only in dermal tissues (epidermis, endodermis and periderm). This protein, previously called GP57, is now referred to as EDGP (E xtracellular D ermal G lyco P rotein). We purified sufficient quantities of EDGP to obtain amino-acid sequences on two internal tryptic peptides and screened a cDNA library of young carrot roots with antiserum to EDGP and with oligonucleotides corresponding to the peptides. Here we report the derived amino-acid sequence of EDGP. Sequence comparisons show that it has 40% amino-acid sequence identity with 7S basic globulin, a protein that is released when soybean seeds are soaked in hot water for a few hours. We suggest that these two proteins belong to a new family of dermal proteins. As far as we know, this is the first reported derived amino-acid sequence for protein that is specific to the epidermis and other dermal tissues. The level of EDGP mRNA is low in dry seeds, but increases rapidly in growing seedlings as they develop dermal tissues. The level of mRNA is low in storage roots, but increases rapidly in response to wounding. The presence of EDGP in dermal tissues and its up-regulation in response to wounding indicate a role in the response of plants to biotic and-or abiotic stresses. An unusual feature of the amino-acid sequence of EDGP is that it contains a short motif, which is present at the active site of aspartyl proteases such as pepsin and chymosin.Abbreviations cDNA
copy DNA
- 2,4-D
2,4-dichlorophen-oxyacetic acid
- EDGP
extracellular dermal glycoprotein
- 7SBG
7S basic globulin
Supported by a contract from the United States Department of Energy (Energy Biosciences) (to M.J.C.) and a Grant-in-Aid for Special Research on Priority Areas (01660002, Cellular and Molecular Basis for Reproductive Processes in Plants) from the Ministry of Education, Science and Culture, and by the Fund from Basic Research Core System of Science and Technology Agency, Japan (to S.S.). 相似文献
12.
Proteins and Carbohydrates in Xylem Sap from Squash Root 总被引:8,自引:0,他引:8
Satoh Shinobu; Iizuka Chika; Kikuchi Akira; Nakamura Norio; Fujii Tadashi 《Plant & cell physiology》1992,33(7):841-847
The xylem sap from squash roots was collected from the cut surfaceof stems, and the proteins and carbohydrates in the sap wereanalyzed. The sap contained 18.6 µg ml1 proteinand the major polypeptides were as follows: 1) two polypeptides,of 75 and 40 kDa, with high-mannose glycans, the levels of whichincreased for about 24 h after cutting and then decreased; 2)a 32-kDa polypeptide, which appeared soon after cutting, disappearedand then reappeared again 4864 h after cutting; and 3)a 19-kDa and a 14-kDa polypeptide, which were present constitutively.The carbohydrates contained in the xylem sap were fractionatedinto 80% ethanol-soluble and -insoluble material, and whichwere analyzed by high-performance liquid chromatography, gaschromatography and enzymatic mathods. The former fraction containedconsiderable amounts of myo-inositol and fructose as free sugarsand oligosaccharides composed mainly of galactose, arabinoseand glucose. The latter contained polysaccharides composed mainlyof uronic acids, galactose and arabinose. The possible significanceof these substances, which may mediate the interactions betweenthe root and the aerial organs, is discussed. (Received April 20, 1992; Accepted July 4, 1992) 相似文献
13.
Tomohiro Kiyosue Jiro Nakayama Shinobu Satoh Akira Isogai Akinori Suzuki Hiroshi Kamada Hiroshi Harada 《Planta》1992,186(3):337-342
ECP31, an embryogenic-cell protein from carrot (Daucus carota L.), was purified by sequential column-chromatographic steps and digested by V8 protease on a nitrocellulose membrane. The resultant peptides were separated by reverse-phased column chromatography and sequenced. The sequences obtained were 70–80% homologous to those of a late-embryogenesis-abundant protein (D34) from cotton (Baker et al, 1988, Plant Mol. Biol. 11, 227–291). The level of ECP31 in somatic embryos of carrot was increased by treatment of the embryos with 3.7 · 10–6 M abscisic acid (ABA) for 48 h, and there was no change in this enhanced level for up to 192 h in the presence of ABA. No similar enhancing effect of ABA was observed on the level of ECP31 in embryogenic callus or segments of carrot hypocotyls. In an immunohistochemical analysis, ECP31 was found in epidermal tissue and in the vascular system of ABA-treated somatic embryos.Abbreviations ABA
abscisic acid
- 2,4-D
2,4-dichlorophenoxyacetic acid
- LEA protein
late-embryogenesis-abundant protein
To whom correspondence should be addressedThis work was supported in part by a grant-in-aid for Special Research in Priority Areas (Project No. 02242102) from the Ministry of Education, Science and Culture, Japan, and by Special Coordination Funds of the Science and Technology Agency of the Japanese Government. 相似文献
14.
15.
Kazuki Nakajima Emi Ito Kazuaki Ohtsubo Ken Shirato Rina Takamiya Shinobu Kitazume Takashi Angata Naoyuki Taniguchi 《Molecular & cellular proteomics : MCP》2013,12(9):2468-2480
Nucleotide sugars are the donor substrates of various glycosyltransferases, and an important building block in N- and O-glycan biosynthesis. Their intercellular concentrations are regulated by cellular metabolic states including diseases such as cancer and diabetes. To investigate the fate of UDP-GlcNAc, we developed a tracing method for UDP-GlcNAc synthesis and use, and GlcNAc utilization using 13C6-glucose and 13C2-glucosamine, respectively, followed by the analysis of mass isotopomers using LC-MS.Metabolic labeling of cultured cells with 13C6-glucose and the analysis of isotopomers of UDP-HexNAc (UDP-GlcNAc plus UDP-GalNAc) and CMP-NeuAc revealed the relative contributions of metabolic pathways leading to UDP-GlcNAc synthesis and use. In pancreatic insulinoma cells, the labeling efficiency of a 13C6-glucose motif in CMP-NeuAc was lower compared with that in hepatoma cells.Using 13C2-glucosamine, the diversity of the labeling efficiency was observed in each sugar residue of N- and O-glycans on the basis of isotopomer analysis. In the insulinoma cells, the low labeling efficiencies were found for sialic acids as well as tri- and tetra-sialo N-glycans, whereas asialo N-glycans were found to be abundant. Essentially no significant difference in secreted hyaluronic acids was found among hepatoma and insulinoma cell lines. This indicates that metabolic flows are responsible for the low sialylation in the insulinoma cells. Our strategy should be useful for systematically tracing each stage of cellular GlcNAc metabolism.Protein glycosylation, which is the most abundant post-translational modification, has important roles in many biological processes by modulating conformation and stability, whereas its dysregulation is associated with various diseases such as diabetes and cancer (1, 2). Glycosylation is regulated by various factors including glucose metabolism, the availability and localization of nucleotide sugars, and the expression and localization of glycosyltransferases (3, 4). Thus, ideally all of these components should be considered when detecting changes in a dynamic fashion; namely, it is necessary not only to take a snapshot but also to make movies of the dynamic changes in glycan metabolism.Glucose is used by living cells as an energy source via the glycolytic pathway as well as a carbon source for various metabolites including nucleotide sugars (e.g. UDP-GlcNAc and CMP-NeuAc). These nucleotide sugars are transported into the Golgi apparatus, and added to various glycans on proteins. UDP-GlcNAc is the donor substrate for N-acetylglucosaminyl (GlcNAc)1 transferases; alternatively, it is used in the cytosol for O-GlcNAc modification (i.e. O-GlcNAcylation) of intracellular proteins (5). The UDP-GlcNAc synthetic pathway is complex as it is a converging point of glucose, nucleotide, fatty acid and amino acid metabolic pathways. Thus, the metabolic flow of glucose modulates the branching patterns of N-glycans via UDP-GlcNAc concentrations because many of the key GlcNAc transferases that determine the branching patterns have widely different Km values for UDP-GlcNAc ranging from 0.04 mm to 11 mm (6, 7). Indeed, it was demonstrated that the branching formation of N-glycans in T cells is stimulated by the supply from the hexosamine pathway, whereby it regulates autoimmune reactions promoted by T cells (8).UDP-GlcNAc is also used for the synthesis of CMP-NeuAc, the donor substrate for sialyltransferases (9). The CMP-NeuAc concentration is controlled by the feedback inhibition of UDP-GlcNAc epimerase/ManNAc kinase by the final product CMP-NeuAc, and hence a high CMP-NeuAc level reduces metabolic flow in CMP-NeuAc de novo synthesis (10). However, there is still only limited information about how the levels of nucleotide sugars dynamically change in response to the environmental cues, and how such changes are reflected in the glycosylation of proteins.Stable isotope labeling is a promising approach to quantify metabolic changes in response to external cues (11, 12). For example, the use of nuclear magnetic resonance to obtain isotopomer signals of metabolically labeled molecules has been applied to trace the flux in glycolysis and fatty acid metabolism (13). An approach based on the mass isotopomers of labeled metabolites with 13C6-glucose has been developed to monitor the UDP-GlcNAc synthetic pathway (13–15). The method based on the labeling ratio of each metabolite related to UDP-GlcNAc synthesis has clarified the contribution of each metabolic pathway (14). Moseley reported a novel deconvolution method for modeling UDP-GlcNAc mass isotopomers (15).Previous studies into the use of nucleotide sugars in glycosylation have relied on the specific detection of metabolically radiolabeled glycans (16). It is possible not only to deduce the glycan structures but also to trace their relative contributions to glycan synthesis without MS. On the other hand, mass isotopomer analysis of glycans labeled with stable isotope provides the ratios of labeled versus unlabeled molecules from MS spectra and structural details of the glycans. However, there are only a limited number of publications reporting the application of stable isotope labeling of glycans for monitoring the dynamics of glycans (17). To date, there have been no reports describing a systematic method for tracing cellular GlcNAc biosynthesis and use based on mass isotopomer analysis.The aim of this study was to extend our knowledge of the synthesis and metabolism of UDP-GlcNAc as well as its use in the synthesis of CMP-NeuAc, N- and O-glycans. We recently developed a conventional HPLC method for simultaneous determination of nucleotide sugars including unstable CMP-NeuAc (18). We first established an LC-MS method for isotopomer analysis of 13C6-glucose labeled nucleotide sugars for tracing UDP-GlcNAc metabolism from synthesis to use, because previous methods were not suitable for estimating UDP-GlcNAc use in CMP-NeuAc de novo synthesis (15). We also established a method for isotopomer analysis of labeled N- and O-glycan to monitor the metabolic flow of hexosamine into glycans. Using these two methods, we demonstrated the differences in the use of hexosamines between hepatoma and pancreatic insulinoma cell lines. Our approach may be useful for identifying a metabolic “bottleneck” that governs the turnover speed and patterns of cellular glycosylation, which may be relevant for various applications including glycoprotein engineering and discovery of disease biomarkers. 相似文献
16.
Sachiko N. Isobe Hideki Hirakawa Shusei Sato Fumi Maeda Masami Ishikawa Toshiki Mori Yuko Yamamoto Kenta Shirasawa Mitsuhiro Kimura Masanobu Fukami Fujio Hashizume Tomoko Tsuji Shigemi Sasamoto Midori Kato Keiko Nanri Hisano Tsuruoka Chiharu Minami Chika Takahashi Tsuyuko Wada Akiko Ono Kumiko Kawashima Naomi Nakazaki Yoshie Kishida Mitsuyo Kohara Shinobu Nakayama Manabu Yamada Tsunakazu Fujishiro Akiko Watanabe Satoshi Tabata 《DNA research》2013,20(1):79-92
17.
Seiji Hayashizaki Shinobu Hirai Yumi Ito Yoshiko Honda Yosefu Arime Ichiro Sora Haruo Okado Tohru Kodama Masahiko Takada 《PloS one》2013,8(10)
Dopamine regulates the psychomotor stimulant activities of amphetamine-like substances in the brain. The effects of dopamine are mediated through five known dopamine receptor subtypes in mammals. The functional relevance of D5 dopamine receptors in the central nervous system is not well understood. To determine the functional relevance of D5 dopamine receptors, we created D5 dopamine receptor-deficient mice and then used these mice to assess the roles of D5 dopamine receptors in the behavioral response to methamphetamine. Interestingly, D5 dopamine receptor-deficient mice displayed increased ambulation in response to methamphetamine. Furthermore, dopamine transporter threonine phosphorylation levels, which regulate amphetamine-induced dopamine release, were elevated in D5 dopamine receptor-deficient mice. The increase in methamphetamine-induced locomotor activity was eliminated by pretreatment with the dopamine transporter blocker GBR12909. Taken together, these results suggest that dopamine transporter activity and threonine phosphorylation levels are regulated by D5 dopamine receptors. 相似文献
18.
Katayama Kazuhiro Hosui Atsushi Sakai Yoshiyuki Itou Minoru Matsuzaki Yasushi Takamori Yoriyuki Hosho Keiko Tsuru Tomomi Takikawa Yasuhiro Michitaka Kojiro Ogawa Eishin Miyoshi Yoko Ito Toshifumi Ida Shinobu Hamada Izumi Miyoshi Katsunori Kodama Hiroko Takehara Tetsuo 《Biological trace element research》2020,195(1):71-81
Biological Trace Element Research - The essential trace element zinc maintains liver functions. Liver diseases can alter overall zinc concentrations, and hypozincemia is associated with various... 相似文献
19.
20.
Tatsuyoshi Kobayashi Yoshio Sakai Shinobu Mutoh 《Bioscience, biotechnology, and biochemistry》2013,77(6):378-386
The purpose of this study is to find optimal conditions for pre-hydrolysis in the new wood saccharification process with strong sulfuric acid. In the experiment, the hydrolysis rate of resistant fraction of pentosan of white birch (Shirakamba, Betula platyphylla Sukatchev var. japonica Hara) wood and the decomposition rate of xylose are measured in acid concentrations ranging from 30 to 60% at temperatures ranging from 30 to 90°C. The hydrolysis of resistant pentosan of white birch and the decomposition of xylose are the first-order reactions. The first-order reaction constant of hydrolysis of resistant pentosan, kB min-1, is expressed by the following empirical equations as the function of percentage concentration of sulfuric acid, C, and reaction temperature described by absolute temperature, T°K, ranging from 40 to 80°C: where sulfuric acid concentrations range from 30 to 50%; where sulfuric acid concentration is 60%.The first-order reaction constant of decomposition of xylose, k2 min-1, is expressed by the following empirical equation as the function of sulfuric acid strength described by acidity function, H0, and reaction temperature described by absolute temperature, T°K, in sulfuric acid concentrations ranging from 30 to 60% at temperatures within the range of 40 to 100°C. where C is sulfuric acid strength described by acidity function, H0. 相似文献