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11.
Sally L. Hanton Loren A. Matheson Laurent Chatre Federica Brandizzi 《The Plant journal : for cell and molecular biology》2009,57(6):963-974
Protein export from the endoplasmic reticulum (ER) is mediated by the accumulation of COPII proteins such as Sar1, Sec23/24 and Sec13/31 at specialized ER export sites (ERES). Although the distribution of COPII components in mammalian and yeast systems is established, a unified model of ERES dynamics has yet to be presented in plants. To investigate this, we have followed the dynamics of fluorescent fusions to inner and outer components of the coat, AtSec24 and AtSec13, in three different plant model systems: tobacco and Arabidopsis leaf epidermis, as well as tobacco BY-2 suspension cells. In leaves, AtSec24 accumulated at Golgi-associated ERES, whereas AtSec13 showed higher levels of cytosolic staining compared with AtSec24. However, in BY-2 cells, both AtSec13 and AtSec24 labelled Golgi-associated ERES, along with AtSec24. To correlate the distribution of the COPII coat with the dynamics of organelle movement, quantitative live-cell imaging analyses demonstrated that AtSec24 and AtSec13 maintained a constant association with Golgi-associated ERES, irrespective of their velocity. However, recruitment of AtSec24 and AtSec13 to ERES, as well as the number of ERES marked by these proteins, was influenced by export of membrane cargo proteins from the ER to the Golgi. Additionally, the increased availability of AtSec24 affected the distribution of AtSec13, inducing recruitment of this outer COPII coat component to ERES. These results provide a model that, in plants, protein export from the ER occurs via sequential recruitment of inner and outer COPII components to form transport intermediates at mobile, Golgi-associated ERES. 相似文献
12.
Stephen LR Ellison Claire A English Malcolm J Burns Jacquie T Keer 《BMC biotechnology》2006,6(1):33-11
Background
Accurate quantification of DNA using quantitative real-time PCR at low levels is increasingly important for clinical, environmental and forensic applications. At low concentration levels (here referring to under 100 target copies) DNA quantification is sensitive to losses during preparation, and suffers from appreciable valid non-detection rates for sampling reasons. This paper reports studies on a real-time quantitative PCR assay targeting a region of the human SRY gene over a concentration range of 0.5 to 1000 target copies. The effects of different sample preparation and calibration methods on quantitative accuracy were investigated. 相似文献13.
The 18S ribosomal RNAs of 21 tetrapods were sequenced and aligned with five
published tetrapod sequences. When the coelacanth was used as an outgroup,
Lissamphibia (living amphibians) and Amniota (amniotes) were found to be
statistically significant monophyletic groups. Although little resolution
was obtained among the lissamphibian taxa, the amniote sequences support a
sister-group relationship between birds and mammals. Portions of the 28S
ribosomal RNA (rRNA) molecule in 11 tetrapods also were sequenced, although
the phylogenetic results were inconclusive. In contrast to previous
studies, deletion or down- weighting of base-paired sites were found to
have little effect on phylogenetic relationships. Molecular evidence for
amniote relationships is reviewed, showing that three genes
(beta-hemoglobin, myoglobin, and 18S rRNA) unambiguously support a
bird-mammal relationship, compared with one gene (histone H2B) that favors
a bird- crocodilian clade. Separate analyses of four other genes (alpha-
crystallin A, alpha-hemoglobin, insulin, and 28S rRNA) and a combined
analysis of all sequence data are inconclusive, in that different groups
are defined in different analyses and none are strongly supported. It is
suggested that until sequences become available from a broader array of
taxa, the molecular evidence is best evaluated at the level of individual
genes, with emphasis placed on those studies with the greatest number of
taxa and sites. When this is done, a bird-mammal relationship is most
strongly supported. When regarded in combination with the morphological
evidence for this association, it must be considered at least as plausible
as a bird-crocodilian relationship.
相似文献
14.
15.
Abbie LA Binch Ashley A Cole Lee M Breakwell Anthony LR Michael Neil Chiverton Alison K Cross Christine L Le Maitre 《Arthritis research & therapy》2014,16(5)
Introduction
The degenerate intervertebral disc (IVD) becomes innervated by sensory nerve fibres, and vascularised by blood vessels. This study aimed to identify neurotrophins, neuropeptides and angiogenic factors within native IVD tissue and to further investigate whether pro-inflammatory cytokines are involved in the regulation of expression levels within nucleus pulposus (NP) cells, nerve and endothelial cells.Methods
Quantitative real-time PCR (qRT-PCR) was performed on 53 human IVDs from 52 individuals to investigate native gene expression of neurotrophic factors and their receptors, neuropeptides and angiogenic factors. The regulation of these factors by cytokines was investigated in NP cells in alginate culture, and nerve and endothelial cells in monolayer using RT-PCR and substance P (SP) protein expression in interleukin-1 (IL-1β) stimulated NP cells.Results
Initial investigation on uncultured NP cells identified expression of all neurotrophins by native NP cells, whilst the nerve growth factor (NGF) receptor was only identified in severely degenerate and infiltrated discs, and brain derived neurotrophic factor (BDNF) receptor expressed by more degenerate discs. BDNF expression was significantly increased in infiltrated and degenerate samples. SP and vascular endothelial growth factor (VEGF) were higher in infiltrated samples. In vitro stimulation by IL-1β induced NGF in NP cells. Neurotropin-3 was induced by tumour necrosis factor alpha in human dermal microvascular endothelial cells (HDMECs). SP gene and protein expression was increased in NP cells by IL-1β. Calcitonin gene related peptide was increased in SH-SY5Y cells upon cytokine stimulation. VEGF was induced by IL-1β and interleukin-6 in NP cells, whilst pleiotrophin was decreased by IL-1β. VEGF and pleiotrophin were expressed by SH-SY5Y cells, and VEGF by HDMECs, but were not modulated by cytokines.Conclusions
The release of cytokines, in particular IL-1β during IVD degeneration, induced significant increases in NGF and VEGF which could promote neuronal and vascular ingrowth. SP which is released into the matrix could potentially up regulate the production of matrix degrading enzymes and also sensitise nerves, resulting in nociceptive transmission and chronic low back pain. This suggests that IL-1β is a key regulatory cytokine, involved in the up regulation of factors involved in innervation and vascularisation of tissues. 相似文献16.
Indispensable membrane trafficking events depend on the activity of conserved small guanosine triphosphatases (GTPases), anchored to individual organelle membranes. In plant cells, it is currently unknown how these proteins reach their correct target membranes and interact with their effectors. To address these important biological questions, we studied two members of the ADP ribosylation factor (ARF) GTPase family, ARF1 and ARFB, which are membrane anchored through the same N-terminal myristoyl group but to different target membranes. Specifically, we investigated how ARF1 is targeted to the Golgi and post-Golgi structures, whereas ARFB accumulates at the plasma membrane. While the subcellular localization of ARFB appears to depend on multiple domains including the C-terminal half of the GTPase, the correct targeting of ARF1 is dependent on two domains: an N-terminal ARF1 domain that is necessary for the targeting of the GTPase to membranes and a core domain carrying a conserved MxxE motif that influences the relative distribution of ARF1 between the Golgi and post-Golgi compartments. We also established that the N-terminal ARF1 domain alone was insufficient to maintain an interaction with membranes and that correct targeting is a protein-specific property that depends on the status of the GTP switch. Finally, an ARF1-ARFB chimera containing only the first 18 amino acids from ARF1 was shown to compete with ARF1 membrane binding loci. Although this chimera exhibited GTPase activity in vitro, it was unable to recruit coatomer, a known ARF1 effector, onto Golgi membranes. Our results suggest that the targeting of ARF GTPases to the correct membranes may not only depend on interactions with effectors but also relies on distinct protein domains and further binding partners on the Golgi surface. 相似文献
17.
The three-substituted dipyridyl ligand bis(3-pyridylmethyl)sulfide (L1) was prepared by the reaction of 3-(chloromethyl)pyridine hydrochloride with thioacetamide under basic conditions. L1 was reacted with CuI to give complexes with 1:2 and 1:1 molar ratios. Crystal structures of [(CuI)2(L1)]∞ (1) and [CuI(L1)]∞ (2) were determined. In complex 1 the CuI species formed a one-dimensional staircase polymer to which L1 was bound in a side-by-side fashion with π-π interactions between the ligands on each side. Complex 2 consisted of a one-dimensional ribbon polymer of metallomacrocycles formed from two L1 ligands bridging Cu2I2 dimers which were fused within the macrocyclic ring. The analogous disulfide ligand bis(3-pyridylmethyl)disulfide (L2) was prepared by oxidation of the corresponding thiol 3-(sulfanylmethyl)pyridine. L2 was reacted with CuI in 1:2 and 1:1 molar ratios and products isolated but only the 1:1 product was able to be crystallised. The crystal structure of [CuI(L2)]∞ (3) consisted of a one-dimensional ribbon polymer of metallomacrocycles formed from two L2 ligands linked through Cu2I2 dimers. The difference in the metallomacrocycle linking between the related structures 2 and 3 was attributed to the difference in ligand conformation. 相似文献
18.
An integrated metabonomic approach to describe temporal metabolic disregulation induced in the rat by the model hepatotoxin allyl formate 总被引:4,自引:0,他引:4
Yap IK Clayton TA Tang H Everett JR Hanton G Provost JP Le Net JL Charuel C Lindon JC Nicholson JK 《Journal of proteome research》2006,5(10):2675-2684
The time-related metabolic events in rat liver, plasma, and urine following hepatotoxic insult with allyl formate (75 mg/kg) were studied using a combination of high-resolution liquid state and magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopic methods together with pattern recognition analysis. The metabonomics results were compared with the results of conventional plasma chemistry and histopathological assessments of liver damage. Various degrees of liver damage were observed in different animals, and this variation was reflected in all of the analyses. Furthermore, each analysis revealed a high degree of functional and structural recovery by the end of the study. The allyl formate-induced changes included hepatocellular necrosis, hepatic lipidosis, decreased liver glycogen and glucose, decreased plasma lipids, increased plasma creatine and tyrosine, increased urinary taurine and creatine, and decreased urinary TCA cycle intermediates. The observed reductions in hepatic glycogen and glucose suggest increased glucose utilization and are consistent with the expected depletion of hepatic ATP following mitochondrial impairment, assuming that there is a consequent increase in energy production from glycolysis. The increase in plasma tyrosine is consistent with impaired protein synthesis, a known consequence of ATP depletion. Partial least squares-based cross-correlation of the variation in the liver and plasma NMR profiles indicated that the allyl formate-induced increase in liver lipids correlated with the decrease in plasma lipids. This suggests disruption in lipid transport from the liver to plasma, which could arise through impaired apolipoprotein synthesis, as with ethionine. 相似文献
19.
Renna L Hanton SL Stefano G Bortolotti L Misra V Brandizzi F 《Plant molecular biology》2005,58(1):109-122
Golgins are a family of coiled-coil proteins that are associated with the Golgi apparatus. They are necessary for tethering events in membrane fusion and may act as structural support for Golgi cisternae. Here we report on the identification of an Arabidopsis golgin which is a homologue of CASP, a known transmembrane mammalian and yeast golgin. Similar to its homologues, the plant CASP contains a long N-terminal coiled-coil region protruding into the cytosol and a C-terminal transmembrane domain with amino acid residues which are highly conserved across species. Through fluorescent protein tagging experiments, we show that plant CASP localizes at the plant Golgi apparatus and that the C-terminus of this protein is sufficient for its localization, as has been shown for its mammalian counterpart. In addition, we demonstrate that the plant CASP is able to localize at the mammalian Golgi apparatus. However, mutagenesis of a conserved tyrosine in the transmembrane domain revealed that it is necessary for ER export and Golgi localization of the Arabidopsis CASP in mammalian cells, but is not required for its correct localization in plant cells. These data suggest that mammalian and plant cells have different mechanisms for concentrating CASP in the Golgi apparatus.†These authors have contributed equally to the work 相似文献
20.
Kate L E Phillips Neil Chiverton Anthony LR Michael Ashley A Cole Lee M Breakwell Gail Haddock Rowena AD Bunning Alison K Cross Christine L Le Maitre 《Arthritis research & therapy》2013,15(6):R213