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171.
Yitao Luo Chengqiang Zhang Li Ma Yuxiao Zhang Zhengyuan Liu Li Chen Rui Wang Yujing Luan Yulan Rao 《Journal of lipid research》2022,63(6):100228
7-dehydrocholesterol (7-DHC) and cholesterol (CHOL) are biomarkers of Smith-Lemli-Opitz Syndrome (SLOS), a congenital autosomal recessive disorder characterized by elevated 7-DHC level in patients. Hair samples have been shown to have great diagnostic and research value, which has long been neglected in the SLOS field. In this study, we sought to investigate the feasibility of using hair for SLOS diagnosis. In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), hair samples were completely pulverized and extracted by micro-pulverized extraction in alkaline solution or in n-hexane. After microwave-assisted derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide, the analytes were measured by GC-MS. We found that the limits of determination for 7-DHC and CHOL were 10 ng/mg and 8 ng/mg, respectively. In addition, good linearity was obtained in the range of 50–4000 ng/mg and 30–6000 ng/mg for 7-DHC and CHOL, respectively, which fully meets the requirement for SLOS diagnosis and related research. Finally, by applying the proposed method to real hair samples collected from 14 healthy infants and two suspected SLOS patients, we confirmed the feasibility of hair analysis as a diagnostic tool for SLOS. In conclusion, we present an optimized and validated analytical method for the simultaneous determination of two SLOS biomarkers using human hair. 相似文献
172.
Metabolic engineering to produce phytochromes with phytochromobilin, phycocyanobilin, or phycoerythrobilin chromophore in Escherichia coli 总被引:3,自引:0,他引:3
By co-expression of heme oxygenase and various bilin reductase(s) in a single operon in conjunction with apophytochrome using two compatible plasmids, we developed a system to produce phytochromes with various chromophores in Escherichia coli. Through the selection of different bilin reductases, apophytochromes were assembled with phytochromobilin, phycocyanobilin, and phycoerythrobilin. The blue-shifted difference spectra of truncated phytochromes were observed with a phycocyanobilin chromophore compared to a phytochromobilin chromophore. When the phycoerythrobilin biosynthetic enzymes were co-expressed, E. coli cells accumulated orange-fluorescent phytochrome. The metabolic engineering of bacteria for the production of various bilins for assembly into phytochromes will facilitate the molecular analysis of photoreceptors. 相似文献
173.
Emma J. Shanks 《Analytical biochemistry》2010,396(2):194-10438
Activity of the pterin- and folate-salvaging enzymes pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). Although this assay has been adequate to study the biology of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymatically coupled screening assay in which the product of the enzymatic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-molecule inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS. 相似文献
174.
Irina F. Sevrioukova 《Journal of molecular biology》2009,390(5):924-11096
Apoptosis-inducing factor (AIF) is a bifunctional mitochondrial flavoprotein critical for energy metabolism and induction of caspase-independent apoptosis, whose exact role in normal mitochondria remains unknown. Upon reduction with NADH, AIF undergoes dimerization and forms tight, long-lived FADH2-NAD charge-transfer complexes (CTC) that are proposed to be functionally important. To obtain a deeper insight into structure/function relations and redox mechanism of this vitally important protein, we determined the X-ray structures of oxidized and NADH-reduced forms of naturally folded recombinant murine AIF. Our structures reveal that CTC with the pyridine nucleotide is stabilized by (i) π-stacking interactions between coplanar nicotinamide, isoalloxazine, and Phe309 rings; (ii) rearrangement of multiple aromatic residues in the C-terminal domain, likely serving as an electron delocalization site; and (iii) an extensive hydrogen-bonding network involving His453, a key residue that undergoes a conformational switch to directly interact with and optimally orient the nicotinamide for charge transfer. Via the His453-containing peptide, redox changes in the active site are transmitted to the surface, promoting AIF dimerization and restricting access to a primary nuclear localization signal through which the apoptogenic form is transported to the nucleus. Structural findings agree with biochemical data and support the hypothesis that both normal and apoptogenic functions of AIF are controlled by NADH. 相似文献
175.
Heinz A Hannemann F Müller JJ Heinemann U Bernhardt R 《Biochemical and biophysical research communications》2005,338(1):491-498
Adrenodoxin (Adx) is a [2Fe-2S] ferredoxin involved in electron transfer reactions in the steroid hormone biosynthesis of mammals. In this study, we deleted the sequence coding for the complete interaction domain in the Adx cDNA. The expressed recombinant protein consists of the amino acids 1-60, followed by the residues 89-128, and represents only the core domain of Adx (Adx-cd) but still incorporates the [2Fe-2S] cluster. Adx-cd accepts electrons from its natural redox partner, adrenodoxin reductase (AdR), and forms an individual complex with this NADPH-dependent flavoprotein. In contrast, formation of a complex with the natural electron acceptor, CYP11A1, as well as electron transfer to this steroid hydroxylase is prevented. By an electrostatic and van der Waals energy minimization procedure, complexes between AdR and Adx-cd have been proposed which have binding areas different from the native complex. Electron transport remains possible, despite longer electron transfer pathways. 相似文献
176.
Proanthocyanidins, also known as condensed tannins, are oligomers or polymers of flavan-3-ol units. In spite of important breakthroughs in our understanding of the biosynthesis of the major building blocks of proanthocyanidins, (+)-catechin and (-)-epicatechin, important questions still remain to be answered as to the exact nature of the molecular species that undergo polymerization, and the mechanisms of assembly. We review the structures of proanthocyanidins reported over the past 12 years in the context of biosynthesis, and summarize the outstanding questions concerning synthesis of proanthocyanidins from the chemical, biochemical and molecular genetic perspectives. 相似文献
177.
Lassing I Schmitzberger F Björnstedt M Holmgren A Nordlund P Schutt CE Lindberg U 《Journal of molecular biology》2007,370(2):331-348
An essential consequence of growth factor-mediated signal transduction is the generation of intracellular H2O2. It operates as a second messenger in the control of actin microfilament dynamics, causing rapid and dramatic changes in the morphology and motile activity of stimulated cells. Little is understood about the molecular mechanisms causing these changes in the actin system. Here, it is shown that H2O2 acts directly upon several levels of this system, and some of the mechanistic effects are detailed. We describe the impact of oxidation on the polymerizability of non-muscle β/γ-actin and compare with that of muscle α-actin. Oxidation of β/γ-actin can cause a complete loss of polymerizability, crucially, reversible by the thioredoxin system. Further, oxidation of the actin impedes its interaction with profilin and causes depolymerization of filamentous actin. The effects of oxidation are critically dependent on the nucleotide state and the concentration of Ca2+. We have determined the crystal structure of oxidized β-actin to a resolution of 2.6 Å. The arrangement in the crystal implies an antiparallel homodimer connected by an intermolecular disulfide bond involving cysteine 374. Our data indicate that this dimer forms under non-polymerizing and oxidizing conditions. We identify oxidation of cysteine 272 in the crystallized actin dimer, likely to a cysteine sulfinic acid. In β/γ-actin, this is the cysteine residue most reactive towards H2O2 in solution, and we suggest plausible structural determinants for its reactivity. No other oxidative modification was obvious in the structure, highlighting the specificity of the oxidation by H2O2. Possible consequences of the observed effects in a cellular context and their potential relevance are discussed. 相似文献
178.
Zhubo DaiGuanghong Cui Shu-Feng Zhou Xianan ZhangLuqi Huang 《Journal of plant physiology》2011,168(2):148-157
The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is a rate-limiting step in the isoprenoid biosynthesis via the MVA pathway. In this study, the full-length cDNA encoding HMGR (designated as SmHMGR2, GenBank accession no. FJ747636) was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE). The cloned gene was then transformed into the hairy root of S. miltiorrhiza, and the enzyme activity and production of diterpenoid tanshinones and squalene were monitored. The full-length cDNA of SmHMGR2 comprises 1959 bp, with a 1653-bp open reading frame encoding a 550-amino-acid protein. Molecular modeling showed that SmHMGR2 is a new HMGR with a spatial structure similar to other plant HMGRs. SmHMGR2 contains two HMG-CoA-binding motifs and two NADP(H)-binding motifs. The SmHMGR2 catalytic domain can form a homodimer. The deduced protein has an isoelectric point of 6.28 and a calculated molecular weight of approximately 58.67 kDa. Sequence comparison analysis showed that SmHMGR2 had the highest homology to HMGR from Atractylodes lancea. As expected, a phylogenetic tree analysis indicates that SmHMGR2 belongs to plant HMGR group. Tissue expression pattern analysis shows that SmHMGR2 is strongly expressed in the leaves, stem, and roots. Functional complementation of SmHMGR2 in HMGR-deficient mutant yeast JRY2394 demonstrates that SmHMGR2 mediates the MVA biosynthesis in yeasts. Overexpression of SmHMGR2 increased enzyme activity and enhanced the production of tanshinones and squalene in cultured hairy roots of S. miltiorrhiza. Our DNA gel blot analysis has confirmed the presence and integration of the associated SmHMGR2 gene. SmHMGR2 is a novel and important enzyme involved in the biosynthesis of diterpenoid tanshinones in S. miltiorrhiza. 相似文献
179.
Effects of exogenous calcium chloride (CaCl2) (20 mM) on photosynthetic gas exchange, photosystem II photochemistry, and the activities of antioxidant enzymes in tobacco plants under high temperature stress (43 °C for 2 h) were investigated. Heat stress resulted in a decrease in net photosynthetic rate (Pn), stomatal conductance as well as the apparent quantum yield (AQY) and carboxylation efficiency (CE) of photosynthesis. Heat stress also caused a decrease of the maximal photochemical efficiency of primary photochemistry (Fv/Fm). On the other hand, CaCl2 application improved Pn, AQY, and CE as well as Fv/Fm under high temperature stress. Heat stress reduced the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD), whereas the activities of these enzymes either decreased less or increased in plants pretreated with CaCl2; glutathione reductase (GR) activity increased under high temperature, and it increased more in plants pretreated with CaCl2. There was an obvious accumulation of H2O2 and O2− under high temperature, but CaCl2 application decreased the contents of H2O2 and O2− under heat stress conditions. Heat stress induced the level of heat shock protein 70 (HSP70), while CaCl2 pretreatment enhanced it. These results suggested that photosynthesis was improved by CaCl2 application in heat-stressed plants and such an improvement was associated with an improvement in stomatal conductance and the thermostability of oxygen-evolving complex (OEC), which might be due to less accumulation of reactive oxygen species. 相似文献
180.
Nitrate reductase activation state in barley roots in relation to the energy and carbohydrate status 总被引:12,自引:0,他引:12
The NADH-dependent nitrate reductase (NR, EC 1.6.6.1) in roots of hydroponically grown barley seedlings was extracted, desalted
and the activity measured in buffer containing either Mg2+ (10 mM) or EDTA (5 mM). The former gives the actual NR activity (NRact) equivalent to dephospho-NR, whereas the latter gives the maximum NR capacity of the dephospho-form (NRmax). Both values together permit an estimation of the NR-phosphorylation state. Changes in NRact and NRmax were followed in response to root aeration or to shoot illumination or shoot removal, and were correlated with sugar contents
and adenylate levels. Ethanol formation was also measured in roots differing in NR activity in order to obtain information
on the relation between anaerobic alcoholic fermentation and nitrate reduction. In aerated roots, NR was highly phosphorylated
(about 80%) and largely inactive. It was partly dephosphorylated (activated) by anoxia or by cellular acidification (pH 4.8
plus propionic acid). Anaerobic activation (dephosphorylation) of NR was stronger at acidic external pH (5) than at slightly
alkaline pH (8), although ATP levels decreased and AMP levels increased at pH 5 and at pH 8 to the same extent. Thus, rapid
changes in the NR-phosphorylation state in response to anaerobiosis were not directly triggered by the adenylate pool, but
rather by cytosolic pH. Under prolonged darkness (24 h) or after shoot removal, NRmax decreased slowly without a large change in the phosphorylation state. This decrease of NRmax was correlated with a large decrease in the sugar content, and was prevented by glucose feeding, which had only minor effects
on the phosphorylation state. Cycloheximide also prevented the decrease in NRmax without affecting the phosphorylation state. In contrast, anaerobiosis or cellular acidification prevented the decrease of
NRmax and at the same time decreased the NR-phosphorylation state. It is suggested that NR turnover in roots is controlled by several
factors: NR synthesis appears to depend on sugar availability, which has little effect on the phosphorylation state; in addition,
NR degradation appears to be strongly affected by the phosphorylation state in such a way that the inactive phospho-NR is
a better substrate for NR degradation than the dephospho-form. The rate of anaerobic ethanol formation was not affected by
NR activity, indicating that the purpose of NR activation under hypoxia or anoxia is not to decrease or prevent alcoholic
fermentation.
Received: 29 August 1996 / Accepted: 8 November 1996 相似文献