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排序方式: 共有111条查询结果,搜索用时 31 毫秒
91.
92.
Tsubasa Shoji Koji Inai Yoshiaki Yazaki Yasutaka Sato Hisabumi Takase Nobukazu Shitan Kazufumi Yazaki Yumi Goto Kiminori Toyooka Ken Matsuoka Takashi Hashimoto 《Plant physiology》2009,149(2):708-718
Nicotine is a major alkaloid accumulating in the vacuole of tobacco (Nicotiana tabacum), but the transporters involved in the vacuolar sequestration are not known. We here report that tobacco genes (NtMATE1 and NtMATE2) encoding transporters of the multidrug and toxic compound extrusion (MATE) family are coordinately regulated with structural genes for nicotine biosynthesis in the root, with respect to spatial expression patterns, regulation by NIC regulatory loci, and induction by methyl jasmonate. Subcellular fractionation, immunogold electron microscopy, and expression of a green fluorescent protein fusion protein all suggested that these transporters are localized to the vacuolar membrane. Reduced expression of the transporters rendered tobacco plants more sensitive to the application of nicotine. In contrast, overexpression of NtMATE1 in cultured tobacco cells induced strong acidification of the cytoplasm after jasmonate elicitation or after the addition of nicotine under nonelicited conditions. Expression of NtMATE1 in yeast (Saccharomyces cerevisiae) cells compromised the accumulation of exogenously supplied nicotine into the yeast cells. The results imply that these MATE-type proteins transport tobacco alkaloids from the cytosol into the vacuole in exchange for protons in alkaloid-synthesizing root cells.Alkaloids are a chemically diverse group of low-molecular weight, nitrogen-containing secondary metabolites with characteristic toxicity and pharmacological activity and may function in the chemical defense of plants against herbivores and pathogens (Facchini, 2001; Steppuhn et al., 2004). Natural hydrophilic products, including alkaloids, are usually stored in the vacuole, which appears to be especially adapted to the bulk storage of chemicals for defensive functions. Due to its nitrogen atom(s), an alkaloid can be protonated and is a base. Because several weakly basic alkaloids, such as nicotine, are present in the lipophilic non-charged form in slightly alkaline solutions, a portion of these alkaloids in the cytoplasm may pass through the tonoplast by simple diffusion. An ion-trap mechanism has been proposed to drive an apparent uphill transport of weakly basic alkaloids against a concentration gradient, in which alkaloids are protonated in the acidic vacuole to become membrane-impermeable hydrophilic molecules (Wink and Roberts, 1998). This trapping mechanism removes transport-competent “free” molecules and thus enables the uphill transport process. As attractive as this model is, it is not known whether and how much the actual vacuolar transport of weakly basic alkaloids depends on the trapping mechanism. In contrast, other alkaloids, which are charged under cytosolic pH conditions, are thought to pass through the tonoplast via a carrier-mediated mechanism (Deus-Newmann and Zenk, 1986; Otani et al., 2005).Nicotine is a major alkaloid synthesized in most commercial varieties of tobacco (Nicotiana tabacum). In tobacco, nicotine is synthesized exclusively in the root and distributed throughout the plant via the xylem, concentrating in the young tissues of aerial parts (Hashimoto and Yamada, 1995; Baldwin, 2001). As much as 60 mm of nicotine accumulates in the vacuoles of the leaf epidermal cells at the tip (Lochmann et al., 2001). Putrescine N-methyltransferase (PMT) catalyzes the first committed step in the nicotine-specific pathway, and a PIP-family reductase, called A622, was also suggested to function in a late step in nicotine biosynthesis (Hibi et al., 1994; Shoji et al., 2000a, 2000b; DeBoer et al., 2009; Kajikawa et al., 2009). PMT and A622 proteins are specifically expressed in the same cell types in the root (Shoji et al., 2000a, 2002). Both enzymes were abundant in the endodermis and cortex cells of the root tips, whereas in the differentiated region of the root, the outermost layer of the cortex and parenchyma cells surrounding the xylem in the vascular bundle contained these proteins. These localization patterns not only substantiated root-specific nicotine biosynthesis but also suggested nicotine synthesis to be intimately associated with the xylem-based transport.Nicotine biosynthesis is positively regulated by the jasmonate-signaling cascade involving the COI1 F-box protein and JAZ repressors (Paschold et al., 2007; Shoji et al., 2008) and by the NIC regulatory loci that specifically control the gene expression of all enzymes known to be involved in the biosynthesis (Legg, 1984; Hibi et al., 1994; Reed and, Jelesko, 2004; Cane et al., 2005; Heim et al., 2007; Katoh et al., 2007). In flavonoid biosynthesis, regulatory genes coordinately regulate not only enzyme genes but also transporter genes responsible for intracellular transport of the metabolites (Koes et al., 2005). In this study, we identified two related tobacco transporters that are coordinately regulated by the NIC loci with nicotine biosynthetic enzymes. Our results suggest that these transporters promote the uptake of nicotine and related alkaloids into the vacuole by using a H+-gradient across the tonoplast in the alkaloid-synthesizing root cells. 相似文献
93.
D. Bernacchi T. Beck-Bunn D. Emmatty Y. Eshed S. Inai J. Lopez V. Petiard H. Sayama J. Uhlig D. Zamir S. Tanksley 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,97(7):1191-1196
Improved-processing tomato lines were produced by the molecular breeding strategy of advanced backcross QTL (AB-QTL) analysis.
These near-isogenic lines (NILs) contained unique introgressions of wild alleles originating from two donor wild species,
Lycopersicon hirsutum (LA1777) and L. pimpinellifolium (LA1589). Wild alleles targeted for trait improvement were selected on the basis of previously published replicated QTL data
obtained from advanced backcross populations for a battery of important agronomic traits. Twenty three NILs were developed
for 15 genomic regions which were predicted to contain 25 quantitative trait factors for the improvement of seven agronomic
traits: total yield, red yield, soluble solids, brix×red yield, viscosity, fruit color, and fruit firmness. An evaluation
of the agronomic performance of the NILs in five locations worldwide revealed that 22 out of the 25 (88%) quantitative factors
showed the phenotypic improvement predicted by QTL analysis of the BC3 populations, as NILs in at least one location. Per-location gains over the elite control ranged from 9% to 59% for brix×red
yield; 14% to 33% for fruit color; 17% to 34% for fruit firmness; 6% to 22% for soluble-solids content; 7% to 22% for viscosity;
15% to 48% for red yield, and 20% to 28% for total yield. The inheritance of QTLs, the implementation of the AB-QTL methodology
for characterizing unadapted germplasm and the applicability of this method to other crops are discussed.
Theor Appl Genet (1998) 97 : 170–180
Received: 27 October 1997 / Accepted: 25 November 1997 相似文献
94.
Iida H Honda Y Matsuyama T Shibata Y Inai T 《Molecular reproduction and development》2006,73(3):342-349
Spetex-1 has recently been isolated by differential display and screening of cDNA library. It encodes a protein of 556 amino acid residues possessing coiled-coil motifs. In the rat seminiferous tubules (ST), Spetex-1 was expressed in the cytoplasm of elongating spermatids. To examine the subcellular distribution of Spetex-1 in mature spermatozoa, we performed biochemical and immunocytochemical approaches. We found that Spetex-1 that was synthesized in the cytoplasm of elongating spermatids was subsequently integrated as a middle piece component into spermatozoa during spermiogenesis. After integration, the majority of Spetex-1 in spermatozoa could be extracted by 6M urea under reduced condition but not released by the treatment of 1% Triton X-100. Immunoelectron microscopy demonstrated that Spetex-1 seemed to locate at the inner side of outer dense fibers (ODFs) in the middle piece or the narrow space between ODFs and axoneme. Spetex-1 might be involved in the stability of the structural complexity comprising axoneme and ODFs in the middle piece of sperm flagellum. 相似文献
95.
Kawauchi D Taniguchi H Watanabe H Saito T Murakami F 《Development (Cambridge, England)》2006,133(6):1113-1123
Nuclei are aggregates of neurons distributed in the central nervous system and are fundamental functional units that share anatomical and physiological features. Despite their importance, the cellular basis that leads to nucleogenesis is only poorly understood. Using exo utero electroporation with an enhanced yellow fluorescent protein (EYFP) gene, we show that the precerebellar neurons derived from the lower rhombic lip (lRL) undergo multiple migration steps to form nuclei. After the unilateral transfer of EYFP to the lRL of embryonic day 12.5 mice, EYFP-labelled neurons migrate tangentially from the lRL in two distinct streams, one towards the ventral metencephalon and the other towards the ventral myelencephalon. These neurons cross the ventral midline and then become radially directed. Labelled neurons in the tangential migratory streams form contralateral clusters in the external cuneate nucleus (ECN) and lateral reticular nucleus (LRN) in the myelencephalon, and bilateral clusters in the pontine grey nucleus (PGN) and reticulotegmental nucleus (RTN) in the metencephalon. Before forming the clusters, EYFP-labelled neurons begin to migrate radially towards the ventricle in close apposition to nestin-positive radial fibres, and then they aggregate as they detach from the fibres. Inhibition of cadherin function in ECN and LRN progenitors caused ipsilateral formation of the ECN and LRN, implying that the transition of their migration from tangential to radial involves a cell-intrinsic mechanism. These observations suggest that nucleogenesis of precerebellar neurons is a result of multi-phasic migration, and that ventricle-directed radial glia-guided migration is a key step for nucleogenesis. 相似文献
96.
An x-ray crystallographic analysis was carried out for Boc-(Aib-DeltaZPhe)4-Aib-OMe (1: Boc = t-butoxycarbonyl; Aib = alpha-aminoisobutyric acid; DeltaZPhe = Z-alpha,beta-didehydrophenylalanine) to provide the precise conformational parameters of the octapeptide segment -(Aib-DeltaZPhe)4-. Peptide 1 adopted a typical 3(10)-helical conformation characterized by = +/-55.8 degrees (50 degrees -65 degrees), = +/-26.7 degrees (15 degrees -45 degrees), and = +/-179.5 degrees (168 degrees -188 degrees) for the average values of the -(Aib-DeltaZPhe)4- segment (the range of the eight values). The 3(10)-helix contains 3.1 residues per turn, being close to the "perfect 3(10)-helix" characterized by 3.0 residues per turn. NMR and Fourier transform infrared (FTIR) spectroscopy revealed that the 3(10)-helical conformation at the atomic resolution is essentially maintained in solution. Energy minimization of peptide 1 by semiempirical molecular orbital calculation converged to a 3(10)-helical conformation similar to the x-ray crystallographic 3(10)-helix. The preference for a 3(10)-helix in the -(Aib-DeltaZPhe)4- segment is ascribed to strong inducers of the 3(10)-helix inherent in Aib and DeltaZPhe residues-in particular, the Aib residues tend to stabilize a 3(10)-helix more effectively. Therefore, the -(Aib-DeltaZPhe)4- segment is useful to rationally design an optically inactive 3(10)-helical backbone, which will be of great importance to provide novel insights into noncovalent and covalent chiral interactions of a helical peptide with a chiral molecule. 相似文献
97.
98.
The noncovalent chiral domino effect (NCDE), defined as chiral interaction upon an N-terminus of a 3(10)-helical peptide, will provide a unique method for structural control of a peptide helix through the use of external chirality. On the other hand, the NCDE has not been considered to be effective for the helicity control of peptides strongly favoring a one-handed screw sense. We here aim to promote the NCDE on peptide helicity using two types of nonapeptides: H-beta-Ala-Delta(Z)Phe-Aib-Delta(Z)Phe-X-(Delta(Z)Phe-Aib)(2)-OCH(3) [Delta(Z)Phe = alpha,beta-didehydrophenylalanine, Aib = alpha-aminoisobutyric acid], where X as the single chirality is L-leucine (1) or L-phenylalanine (2). NMR, IR, and CD spectroscopy as well as energy calculation revealed that both peptides alone form a right-handed 3(10)-helix. The original CD amplitudes or signs in chloroform, irrespective of a strong screw-sense preference in the central chirality, responded sensitively to external chiral information. Namely added Boc-L-amino acid stabilized the original right-handed helix, while the corresponding d-isomer destabilized it or transformed it into a left-handed helix. These peptides were also shown to bind more favorably to an L-isomer from the racemate. Although similar helicity control was observed for analogous nonapeptides bearing an N-terminal Aib residue (Inai, Y.; et al. Biomacromolecules 2003, 4, 122), the present findings demonstrate that the N-terminal replacement by the beta-Ala residue significantly improves the previous NCDE to achieve more effective control of helicity. Semiempirical molecular orbital calculations on complexation of peptide 2 with Boc-(L or D)-Pro-OH reasonably explained the unique conformational change induced by external chirality. 相似文献
99.
The T cell cytokines profoundly modify the phenotypic and functional characteristics of keratinocytes. Until now, no study has focused on the effect of Th1 and Th2 cytokines on keratinocyte permeability. Using a two-layer well culturing system, permeability was assessed through cultured keratinocyte sheet in the presence or absence of various concentrations of IFN-gamma and IL-4. Transepithelial electrical resistance (TER) and the flux of 40 kDa FITC-dextrans were measured across the cultured keratinocyte sheet. IFN-gamma significantly increased the TER in a dose- and time-dependent manner, suggesting that IFN-gamma profoundly inhibited the permeability of ions through the keratinocyte sheet. In contrast, IL-4 did not affect the TER. When compared to medium control, the flux of FITC-dextran of the IFN-gamma group was significantly decreased in a dose-dependent fashion. In sharp contrast, the flux of FITC-dextran was significantly and dose-dependently increased in the presence of IL-4. A significant increase in TER and a significant decrease in the flux of dextran suggested that IFN-gamma clearly reduced the permeability of both ions and high molecular weight material through the keratinocyte sheet. Although IL-4 did not affect the permeability of the ions, it significantly enhanced the permeability of high molecular weight material. A flow cytometric assay revealed that the expression of desmoglein-3 was suppressed by IL-4, but was enhanced by IFN-gamma. The reciprocal regulation of permeability of the cultured keratinocyte sheet by IFN-gamma and IL-4 may be partly related to the modification of intercellular adhesion molecules. 相似文献
100.
A new synthetic route to (E)-beta-phenyl-alpha,beta-dehydroalanine (delta(E)Phe)-containing peptide was presented via photochemical isomerization of the corresponding (Z)-beta-phenyl-alpha,beta-dehydroalanine (delta(Z)Phe)-containing peptide. By applying this method to Boc-Ala-delta(Z)Phe-Val-OMe (Z-I: Boc, t-butoxycarbonyl; OMe, methoxy), Boc-Ala-delta(E)Phe-Val-OMe (E-I) was obtained. The identification of peptide E-I was evidenced by 1H-nmr, 13C-nmr, and uv absorption spectroscopy, elemental analysis, and hydrogenation. The conformation of peptide E-I in CDCl3 was investigated by 1H-nmr spectroscopy (solvent dependence of NH chemical shift and difference nuclear Overhauser effect). Interestingly, peptide E-I differed from peptide Z-I in the hydrogen-bonding mode. Namely, for peptide Z-I, only Val NH participates in intramolecular hydrogen bonding, which leads to a type II beta-turn conformation supported by hydrogen bonding between CO(Boc) and NH(Val). On the other hand, for peptide E-I, two NHs, delta(E)Phe NH and Val NH, participate in intramolecular hydrogen bonding. In both peptides, a remarkable NOE (approximately 11-13%) was observed for Ala C(alpha) H-deltaPhe NH pair. Based on the nmr data and conformational energy calculation, it should be concluded that peptide E-I takes two consecutive gamma-turn conformations supported by hydrogen bonding between CO(Boc) and NH(delta(E)Phe), and between CO(Ala) and NH(Val) as its plausible conformation. 相似文献