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81.
A coumaroyl-ester-3-hydroxylase insertion mutant reveals the existence of nonredundant meta-hydroxylation pathways and essential roles for phenolic precursors in cell expansion and plant growth 总被引:6,自引:0,他引:6
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Abdulrazzak N Pollet B Ehlting J Larsen K Asnaghi C Ronseau S Proux C Erhardt M Seltzer V Renou JP Ullmann P Pauly M Lapierre C Werck-Reichhart D 《Plant physiology》2006,140(1):30-48
Cytochromes P450 monooxygenases from the CYP98 family catalyze the meta-hydroxylation step in the phenylpropanoid biosynthetic pathway. The ref8 Arabidopsis (Arabidopsis thaliana) mutant, with a point mutation in the CYP98A3 gene, was previously described to show developmental defects, changes in lignin composition, and lack of soluble sinapoyl esters. We isolated a T-DNA insertion mutant in CYP98A3 and show that this mutation leads to a more drastic inhibition of plant development and inhibition of cell growth. Similar to the ref8 mutant, the insertion mutant has reduced lignin content, with stem lignin essentially made of p-hydroxyphenyl units and trace amounts of guaiacyl and syringyl units. However, its roots display an ectopic lignification and a substantial proportion of guaiacyl and syringyl units, suggesting the occurrence of an alternative CYP98A3-independent meta-hydroxylation mechanism active mainly in the roots. Relative to the control, mutant plantlets produce very low amounts of sinapoyl esters, but accumulate flavonol glycosides. Reduced cell growth seems correlated with alterations in the abundance of cell wall polysaccharides, in particular decrease in crystalline cellulose, and profound modifications in gene expression and homeostasis reminiscent of a stress response. CYP98A3 thus constitutes a critical bottleneck in the phenylpropanoid pathway and in the synthesis of compounds controlling plant development. CYP98A3 cosuppressed lines show a gradation of developmental defects and changes in lignin content (40% reduction) and structure (prominent frequency of p-hydroxyphenyl units), but content in foliar sinapoyl esters is similar to the control. The purple coloration of their leaves is correlated to the accumulation of sinapoylated anthocyanins. 相似文献
82.
Dayanithi G Mechaly I Viero C Aptel H Alphandery S Puech S Bancel F Valmier J 《Cell calcium》2006,39(3):237-246
Changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) control the setting up of the neuro-muscular synapse in vitro and probably in vivo. Dissociated cultures of purified embryonic (E15) rat motoneurons were used to explore the molecular mechanisms by which endoplasmic reticulum Ca(2+) stores, via both ryanodine-sensitive and IP(3)-sensitive intracellular Ca(2+) channels control [Ca(2+)](i) homeostasis in these neurons during ontogenesis. Fura-2 microspectrofluorimetry monitorings in single neurons showed that caffeine-induced responses of [Ca(2+)](i) increased progressively from days 1-7 in culture. These responses were blocked by ryanodine and nicardipine but not by omega-conotoxin-GVIA or omega-conotoxin-MVIIC suggesting a close functional relationship between ryanodine-sensitive and L-type Ca(v)1 Ca(2+) channels. Moreover, after 6 days in vitro, neurons exhibited spontaneous or caffeine-induced Ca(2+) oscillations that were attenuated by nicardipine. In 1-day-old neurons, both thapsigargin or CPA, which deplete Ca(2+) stores from the endoplasmic reticulum, induced an increase in [Ca(2+)](i) in 75% of the neurons tested. The number of responding motoneurons declined to 25% at 5-6 days in vitro. Xestospongin-C, a membrane-permeable IP(3) receptor inhibitor blocked the CPA-induced [Ca(2+)](i) response in all stages. RT-PCR studies investigating the expression pattern of RYR and IP(3) Ca(2+) channels isoforms confirmed the presence of their different isoforms and provided evidence for a specific pattern of development for RYR channels during the first week in vitro. Taken together, present results show that the control of motoneuronal [Ca(2+)](i) homeostasis is developmentally regulated and suggest the presence of an intracellular ryanodine-sensitive Ca(2+) channel responsible for a Ca(2+)-induced Ca(2+) release in embryonic motoneurons following voltage-dependent Ca(2+) entry via L-type Ca(2+) channels. 相似文献
83.
84.
Hiam A Sebastien D George B Arlette F Kalil J Le Pape P 《Journal of enzyme inhibition and medicinal chemistry》2006,21(3):305-312
A new family of antimicrotubule drugs named (3-haloacetamidobenzoyl) ureas and ethyl 3-haloacetamidobenzoates were found to be cytotoxic to the Leishmania parasite protozoa. While the benzoylureas were shown to strongly inhibit in vitro mammalian brain microtubule assembly, the ethyl ester derivatives were characterized as very poor inhibitors of this process. Ethyl 3-chloroacetamidobenzoate, MF29, was found to be the most efficient drug on the promastigote stage of three Leishmania species (IC50: 0.3-1.8 microM). MF29 maintained its activity against the clinical relevant intracellular stage of L. mexicana with IC50 value of 0.33 microM. It was the only compound that exhibits a high activity on all the Leishmania species tested. This compound appeared to alter parasite microtubule organisation as demonstrated by using antibodies directed against microtubule components and more precisely the class of microtubule decorated by the MAP2-like protein. It is interesting to notice that this MAP2-like protein was identified for the first time in a Leishmania parasite 相似文献
85.
Siddiqui N Tempel W Nedyalkova L Volpon L Wernimont AK Osborne MJ Park HW Borden KL 《Journal of molecular biology》2012,415(5):781-792
The eukaryotic translation initiation factor eIF4E plays key roles in cap-dependent translation and mRNA export. These functions rely on binding the 7-methyl-guanosine moiety (5'cap) on the 5'-end of all mRNAs. eIF4E is regulated by proteins such as eIF4G and eIF4E binding proteins (4EBPs) that bind the dorsal surface of eIF4E, distal to the cap binding site, and modulate cap binding activity. Both proteins increase the affinity of eIF4E for 5'cap. Our understanding of the allosteric effects and structural underpinnings of 4EBP1 or eIF4G binding can be advanced by obtaining structural data on cap-free eIF4E bound to one of these proteins. Here, we report the crystal structure of apo-eIF4E and cap-free eIF4E in complex with a 4EBP1 peptide. We also monitored 4EBP1 binding to cap-free eIF4E in solution using NMR. Together, these studies suggest that 4EBP1 transforms eIF4E into a cap-receptive state. NMR methods were also used to compare the allosteric routes activated by 4EBP1, eIF4G, and the arenavirus Z protein, a negative regulator of cap binding. We observed chemical shift perturbation at the dorsal binding site leading to alterations in the core of the protein, which were ultimately communicated to the unoccupied cap binding site of eIF4E. There were notable similarities between the routes taken by 4EBP1 and eIF4G and differences from the negative regulator Z. Thus, binding of 4EBP1 or eIF4G allosterically drives alterations throughout the protein that increase the affinity of eIF4E for the 5'cap. 相似文献
86.
Liang G Aldous S Merriman G Levell J Pribish J Cairns J Chen X Maignan S Mathieu M Tsay J Sides K Rebello S Whitely B Morize I Pauls HW 《Bioorganic & medicinal chemistry letters》2012,22(2):1049-1054
A solid phase combinatorial library was designed based on X-ray structures and in-silico models to explore an inducible S4+ pocket, which is formed by a simple side-chain rotation of Tyr95. This inducible S4+ pocket is unique to β-tryptase and does not exist for other trypsin-like serine proteases of interest. Therefore, inhibitors utilizing this pocket have inherent advantages for being selective against other proteases in the same family. A member of this library was found to be a potent and selective β-tryptase inhibitor with a suitable pharmacokinetic profile for further clinical evaluation. 相似文献
87.
Sebastien Gagneux 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2012,367(1590):850-859
Tuberculosis (TB) is a disease of antiquity. Yet TB today still causes more adult deaths than any other single infectious disease. Recent studies show that contrary to the common view postulating an animal origin for TB, Mycobacterium tuberculosis complex (MTBC), the causative agent of TB, emerged as a human pathogen in Africa and colonized the world accompanying the Out-of-Africa migrations of modern humans. More recently, evolutionarily ‘modern’ lineages of MTBC expanded as a consequence of the global human population increase, and spread throughout the world following waves of exploration, trade and conquest. While epidemiological data suggest that the different phylogenetic lineages of MTBC might have adapted to different human populations, overall, the phylogenetically ‘modern’ MTBC lineages are more successful in terms of their geographical spread compared with the ‘ancient’ lineages. Interestingly, the global success of ‘modern’ MTBC correlates with a hypo-inflammatory phenotype in macrophages, possibly reflecting higher virulence, and a shorter latency in humans. Finally, various human genetic variants have been associated with different MTBC lineages, suggesting an interaction between human genetic diversity and MTBC variation. In summary, the biology and the epidemiology of human TB have been shaped by the long-standing association between MTBC and its human host. 相似文献
88.
Sebastien Gallien Elodie Duriez Catharina Crone Markus Kellmann Thomas Moehring Bruno Domon 《Molecular & cellular proteomics : MCP》2012,11(12):1709-1723
There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. To date protein quantification in biological samples has been routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), and two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved so as to reliably discriminate the targeted analytes from background interferences. High resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring) and in MS/MS mode (parallel reaction monitoring). The ability of the quadrupole to select a restricted m/z range allows one to overcome the dynamic range limitations associated with trapping devices, and the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performance in terms of selectivity, dynamic range, and sensitivity. This high performance is further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60-min experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein quantification methods in complex samples and address the pressing demand of systems biology or biomarker evaluation studies.Shotgun proteomics has emerged over the past decade as the most effective method for the qualitative study of complex proteomes (i.e., the identification of the protein content), as illustrated by a wealth of publications (1, 2). In this approach, after enzymatic digestion of the proteins, the generated peptides are analyzed by means of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)1 in a data dependent mode. However, the complexity of the digested proteomes under investigation and the wide range of protein abundances limit the reproducibility and the sensitivity of this stochastic approach (3), which is critical if one aims at the systematic quantification of the proteins. Thus, alternative MS approaches have emerged for the systematic quantitative study of complex proteomes, the MS-based targeted proteomics (4). In this hypothesis-driven approach, only specific subsets of analytes (a few targeted peptides used as surrogates for the proteins of interest) are selectively measured in predefined m/z ranges and retention time windows, which overcomes the bias toward most abundant compounds commonly observed with shotgun proteomics. When applied to complex biological samples—for example, bodily fluids such as urine or plasma—targeted proteomics requires high performance instruments allowing measurements of a wide dynamic range (many orders of magnitude), with high sensitivity in order to detect peptides in the low amol range and sufficient selectivity to cope with massive biochemical background (5). Selected reaction monitoring (SRM) on triple quadrupole (6) or triple quadrupole-linear ion trap mass spectrometers (7) has emerged as a means to conduct such analyses (8). Initially applied in the MS analysis of small molecules (9, 10), SRM has gradually emerged as the reference quantitative technique for analyzing proteins (or peptides) in biological samples. When coupled with the isotope dilution strategy (11, 12), this very effective technique allows the precise quantification of proteins (13–18). However, despite the increased selectivity provided by the two-stage mass filtering of SRM (at the precursor and fragment ion levels), the low resolution of mass selection does not allow the systematic removal of interferences (19, 20). Moreover, in proteomics, the biochemical background has a composition similar to that of the analytes of interest, which remains a major hurdle limiting the sensitivity of assays, especially in a bodily fluid matrix. High resolution/accurate mass (HR/AM) analysis represents a promising alternative approach that might more efficiently distinguish the compounds of interest from interferences in targeted proteomics. Such analyses can be conducted on orbitrap-based mass spectrometers because of their high sensitivity and high mass accuracy capabilities (21). The introduction of the benchtop standalone orbitrap mass spectrometer (Exactive) (22) further strengthened the attractiveness of the approach, especially in the field of small molecule analysis (23, 24). However, as quantification using trapping devices intrinsically suffers from a limited dynamic range because of the overall ion capacity, the complexity of biological samples remains very challenging even with the HR/AM approach (25). Targeted protein analysis with triple quadrupole mass spectrometers keeps on showing significant superiority for such samples.2 The recently developed quadrupole-orbitrap mass spectrometer (Q-Exactive) can potentially address this issue.3 It is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection (26, 27). This configuration combines advantages of triple quadrupole instruments for mass filtering and orbitrap-based mass spectrometers for HR/AM measurement. The ability of the instrument to select a restricted m/z range or (sequentially) a small number of precursor ions offers new opportunities for quantification in complex samples by selectively enriching low abundant components. The resulting data, acquired in the so-called single ion monitoring (SIM) mode, fully benefit from the trapping capability while keeping a high acquisition rate as a result of the fast switching time between targeted precursor ions of the quadrupole. Although this mode of data acquisition is possible with a configuration combining a linear ion trap with the orbitrap (as in the LTQ-Orbitrap mass spectrometer), its effectiveness is far more limited in this case. The quadrupole-orbitrap configuration presents significant benefits by selectively isolating a narrow population of precursor ions. Other features of the instrument include its multiplexed trapping capability (26) using either the C-trap or the higher energy collisional dissociation (HCD) cell (28, 29), which opens new avenues in the design of innovative acquisition methods for quantification studies. For the first time, a panel of acquisition methods is designed and applied to targeted quantification at the MS and MS/MS levels. In the latter case, the simultaneous monitoring of multiple MS/MS fragmentation channels, also called parallel reaction monitoring4 (PRM), is particularly promising for quantifying large sets of peptides with increased selectivity. 相似文献
89.
Zhu H Lee HY Tong Y Hong BS Kim KP Shen Y Lim KJ Mackenzie F Tempel W Park HW 《PloS one》2012,7(3):e33943
Kinesin-1 transports various cargos along the axon by interacting with the cargos through its light chain subunit. Kinesin light chains (KLC) utilize its tetratricopeptide repeat (TPR) domain to interact with over 10 different cargos. Despite a high sequence identity between their TPR domains (87%), KLC1 and KLC2 isoforms exhibit differential binding properties towards some cargos. We determined the structures of human KLC1 and KLC2 tetratricopeptide repeat (TPR) domains using X-ray crystallography and investigated the different mechanisms by which KLCs interact with their cargos. Using isothermal titration calorimetry, we attributed the specific interaction between KLC1 and JNK-interacting protein 1 (JIP1) cargo to residue N343 in the fourth TRP repeat. Structurally, the N343 residue is adjacent to other asparagines and lysines, creating a positively charged polar patch within the groove of the TPR domain. Whereas, KLC2 with the corresponding residue S328 did not interact with JIP1. Based on these finding, we propose that N343 of KLC1 can form "a carboxylate clamp" with its neighboring asparagine to interact with JIP1, similar to that of HSP70/HSP90 organizing protein-1's (HOP1) interaction with heat shock proteins. For the binding of cargos shared by KLC1 and KLC2, we propose a different site located within the groove but not involving N343. We further propose a third binding site on KLC1 which involves a stretch of polar residues along the inter-TPR loops that may form a network of hydrogen bonds to JIP3 and JIP4. Together, these results provide structural insights into possible mechanisms of interaction between KLC TPR domains and various cargo proteins. 相似文献
90.
Colado JC Garcia-Masso X Triplett TN Flandez J Borreani S Tella V 《Journal of strength and conditioning research / National Strength & Conditioning Association》2012,26(11):3018-3024
ABSTRACT: Colado JC, Garcia-Masso X, Triplett TN, Flandez J, Borreani S, Tella V. Concurrent validation of the OMNI-Resistance Exercise Scale of perceived exertion with Thera-Band resistance bands. J Strength Cond Res 26(11): 3018-3024, 2012-The concurrent validity of the OMNI-Resistance Exercise Scale (OMNI-RES) of perceived exertion for use with elastic bands was studied during isotonic resistance exercises. Twenty healthy, physically active subjects completed both familiarization and testing sessions. The criterion variables were myoelectric activity, recorded by electromyography, and heart rate, recorded by a heart rate monitor. The subjects performed 2 separate sets of 15 repetitions in each of the 2 testing sessions and for each of the exercises applied (i.e., frontal and lateral raises). One set was carried out with the separation between the hands gripping the elastic band allowing that 15 repetition maximum to be performed in the selected exercise, whereas the other set was carried out with the separation between hands at +50% of the previous grip. The perceived exertion rating for the active muscles and for the overall body, muscular activity, and heart rate were measured during the final repetition of each set. The results showed significant differences (p ≤ 0.001) in myoelectric activity, heart rate, and OMNI-RES scores between the low- and high-intensity sets and the intraclass correlation coefficient was 0.72-0.76. So it can be concluded that the OMNI-RES can be used for monitoring the intensity of exercises when elastic bands are used. This would allow the training stimulus dosage to be precisely controlled in both the session in progress and between different sessions, and allowing to differentiate between different levels of intensity according to the physical aptitudes and special physiological needs of the subjects. 相似文献