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81.
Akira Isogai Akinori Suzuki Shizuo Higashkawa Shimpei Kuyama Saburo Tamura 《Bioscience, biotechnology, and biochemistry》2013,77(12):3033-3035
( + )-α-Kainic acid (1) was synthesized by starting from a building block, N-Boc-3-acetoxyallylglycine ethyl ester (2). The key intermediate, a methyl 4-[(tert-butoxycarbonyl)prenylamino]-5-hydroxy-2-pentenoate derivative (9), was prepared from 2 in eight synthetic steps. After converting 10 into a methyl ester (11), intramolecular ene-carbocyclization of 11 gave a pyrrolidine derivative (12), which was converted to 1 in a moderate yield. 相似文献
82.
《Free radical research》2013,47(2):82-88
AbstractEndothelial dysfunction characterized by decreased nitric oxide (NO) bioavailability is the first stage of coronary artery disease. It is known that one of the factors associated with an increased risk of coronary artery disease is a high plasma level of uric acid. However, causative associations between hyperuricaemia and cardiovascular risk have not been definitely proved. In this work, we tested the effect of uric acid on endothelial NO bioavailability. Electrochemical measurement of NO production in acetylcholine-stimulated human umbilical endothelial cells (HUVECs) revealed that uric acid markedly decreases NO release. This finding was confirmed by organ bath experiments on mouse aortic segments. Uric acid dose-dependently reduced endothelium-dependent vasorelaxation. To reveal the mechanism of decreasing NO bioavailability we tested the effect of uric acid on reactive oxygen species production by HUVECs, on arginase activity, and on acetylcholine-induced endothelial NO synthase phosphorylation. It was found that uric acid increases arginase activity and reduces endothelial NO synthase phosphorylation. Interestingly, uric acid significantly increased intracellular superoxide formation. In conclusion, uric acid decreases NO bioavailability by means of multiple mechanisms. This finding supports the idea of a causal association between hyperuricaemia and cardiovascular risk. 相似文献
83.
Yuzuru Itoh Markus J. Bröcker Shun-ichi Sekine Dieter Söll Shigeyuki Yokoyama 《Journal of molecular biology》2014
The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins and is synthesized on its specific tRNA (tRNASec). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNASec, formed by seryl-tRNA synthetase, to Sec-tRNASec. SelA, a member of the fold-type-I pyridoxal 5′-phosphate-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500 kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNASec revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNASec. The SelA catalytic site is close to the dimer–dimer interface, but the significance of the dimer pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of “depentamerized” SelA variants with mutations at the dimer–dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I pyridoxal 5′-phosphate-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures. 相似文献
84.
The ε-subunit of ATP-synthase is an endogenous inhibitor of the hydrolysis activity of the complex and its α-helical C-terminal domain (εCTD) undergoes drastic changes among at least two different conformations. Even though this domain is not essential for ATP synthesis activity, there is evidence for its involvement in the coupling mechanism of the pump. Recently, it was proposed that coupling of the ATP synthase can vary as a function of ADP and Pi concentration. In the present work, we have explored the possible role of the εCTD in this ADP- and Pi-dependent coupling, by examining an εCTD-lacking mutant of Escherichia coli. We show that the loss of Pi-dependent coupling can be observed also in the εCTD-less mutant, but the effects of Pi on both proton pumping and ATP hydrolysis were much weaker in the mutant than in the wild-type. We also show that the εCTD strongly influences the binding of ADP to a very tight binding site (half-maximal effect ≈ 1 nM); binding at this site induces higher coupling in EFOF1 and increases responses to Pi. It is proposed that one physiological role of the εCTD is to regulate the kinetics and affinity of ADP/Pi binding, promoting ADP/Pi-dependent coupling. 相似文献
85.
The methylerythritol phosphate (MEP) pathway of Plasmodium falciparum (P. falciparum) has become an attractive target for anti-malarial drug discovery. This study describes a kinetic model of this pathway, its use in validating 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) as drug target from the systemic perspective, and additional target identification, using metabolic control analysis and in silico inhibition studies. In addition to DXR, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) can be targeted because it is the first enzyme of the pathway and has the highest flux control coefficient followed by that of DXR. In silico inhibition of both enzymes caused large decrement in the pathway flux. An added advantage of targeting DXS is its influence on vitamin B1 and B6 biosynthesis. Two more potential targets, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, were also identified. Their inhibition caused large accumulation of their substrates causing instability of the system. 相似文献
86.
Huang Z Hoffmann FW Fay JD Hashimoto AC Chapagain ML Kaufusi PH Hoffmann PR 《The Journal of biological chemistry》2012,287(7):4492-4502
Immune complexes composed of IgG-opsonized pathogens, particles, or proteins are phagocytosed by macrophages through Fcγ receptors (FcγRs). Macrophages primed with IFNγ or other pro-inflammatory mediators respond to FcγR engagement by secreting high levels of cytokines and nitric oxide (NO). We found that unprimed macrophages produced lower levels of NO, which required efficient calcium (Ca(2+)) flux as demonstrated by using macrophages lacking selenoprotein K, which is required for FcγR-induced Ca(2+) flux. Thus, we further investigated the signaling pathways involved in low output NO and its functional significance. Evaluation of inducible, endothelial, and neuronal nitric-oxide synthases (iNOS, eNOS, and nNOS) revealed that FcγR stimulation in unprimed macrophages caused a marked Ca(2+)-dependent increase in both total and phosphorylated nNOS and slightly elevated levels of phosphorylated eNOS. Also activated were three MAP kinases, ERK, JNK, and p38, of which ERK activation was highly dependent on Ca(2+) flux. Inhibition of ERK reduced both nNOS activation and NO secretion. Finally, Transwell experiments showed that FcγR-induced NO functioned to increase the phagocytic capacity of other macrophages and required both NOS and ERK activity. The production of NO by macrophages is conventionally attributed to iNOS, but we have revealed an iNOS-independent receptor/enzyme system in unprimed macrophages that produces low output NO. Under these conditions, FcγR engagement relies on Ca(2+)-dependent ERK phosphorylation, which in turn increases nNOS and, to a lesser extent, eNOS, both of which produce low levels of NO that function to promote phagocytosis. 相似文献
87.
目的:克隆并分析绞股蓝法呢基焦磷酸合酶(FPS)基因的全长序列。方法:参照罗汉果法呢基焦磷酸合酶基因,设计扩增绞股蓝FPS基因的3′RACE引物,采用3'RACE和5'RACE法克隆绞股蓝FPS基因全长cDNA。结果:获得绞股蓝FPS基因全长cDNA序列共1288个核苷酸,包含一个1026核苷酸的开放读框,编码342个氨基酸残基,推断该蛋白的相对分子质量为3.94×104。NCBI Blast结果显示绞股蓝FPS基因编码蛋白的氨基酸序列与已知的植物FPS氨基酸序列的同源性为91%~74%,核酸序列的同源性为88%~78%。结论:克隆了绞股蓝FPS基因全长cDNA序列,为进一步研究绞股蓝FPS基因的表达及三萜皂苷合成通路关键酶分子的进化奠定了基础。 相似文献
88.
The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively) are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3). To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney)-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 agonists bromocriptine and quinpirole significantly induced Akt and GSK3 phosphorylation, as well as ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. The D2S receptor-induced Akt signals were profoundly inhibited by the internalization blockers monodansyl cadaverine and concanavalin A. Activation of the D2S receptor in HEK-293/rD2S cells appeared to trigger Akt/phospho-Akt translocation to the cell membrane. In addition to our cell culture experiments, we studied D2 receptor-dependent Akt in vivo by systemic administration of the D2/D3 agonist quinpirole. The results show that quinpirole evoked Akt-Ser473 phosphorylation in the ventral striatum. Furthermore, intra-accumbens administration of wortmannin, a PI3K (phosphoinositide 3-kinase) inhibitor, significantly suppressed the quinpirole-evoked behavioural activation. Overall, we demonstrate that activation of the dopamine D2S receptor stimulates Akt/GSK3 signalling. In addition, in vivo Akt activity in the ventral striatum appears to play an important role in systemic D2/D3 agonist-induced behavioural activation. 相似文献
89.
Nobuhiro Inoue Takafumi Iwasa Kohji Fukunaga Yasuhiko Matsukado Eishichi Miyamoto 《Journal of neurochemistry》1987,48(3):981-988
Glycogen synthase was partially purified from canine brain to about 70% purity. The purified enzyme showed differences from the properties of the skeletal muscle enzyme with respect to molecular weights of the holoenzyme and subunit and phosphopeptide mapping. The multifunctional calmodulin-dependent protein kinase from the brain phosphorylated brain glycogen synthase with concomitant inactivation of the enzyme. Although about 1.3 mol of phosphate/mol subunit was maximally incorporated into glycogen synthase, 0.4 mol of phosphate/mol subunit was sufficient for the maximal inactivation of the enzyme. The results indicate that brain glycogen synthase is regulated in a calmodulin-dependent manner similarly to the skeletal muscle enzyme, but that the brain enzyme is different from the skeletal muscle enzyme. 相似文献
90.
鸭肝脂肪酸合成酶的胍变性与失活 总被引:3,自引:0,他引:3
报道了鸭肝脂肪酸俣成酶在胍变性过程中构变性过程中构象变化和活性变化的关系,首次验证了邹承鲁提出的酶活性部位构象的理论适用于多功能复合酶,同时该酶变性及复性均可测定出多个阶段,且证明有无活性稳态酶存在,在低浓度盐酸胍溶液中该酶的全反应活性和其中两个还原部位单独活性被同步可逆抑制,随着胍浓度增高,出现不可逆失活且程度和速度均迅速提高,在0.54mol/L胍中该酶全反应活性在1.5分种内已有一半不可逆失 相似文献