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Don Trinh Nguyen Jens Christian G?pfert Nobuhiro Ikezawa Gillian MacNevin Meena Kathiresan Jürgen Conrad Otmar Spring Dae-Kyun Ro 《The Journal of biological chemistry》2010,285(22):16588-16598
Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature. 相似文献
3.
A simple and rapid method is described for the purification of supercoiled PM2 DNA by affinity chromatography on columns of H1 histone covalently coupled to agarose. The method does not require the use of intercalating agents or ultracentrifugation procedures. Under the conditions most appropriate for purification, elution is carried out in a single step with buffered 0.7 M NaCl after the sample has been loaded onto the column in buffered 0.2 M NaCl. The DNA eluted at the higher salt concentration consists of supercoiled closed circular DNA at greater than 90% purity independently of the ratio of supercoiled to nicked circular DNA in the input mixture. 相似文献
4.
The lateral intercellular spaces (LIS) of MDCK cell epithelia grown on permeable supports are about 0.4 pH units acidic to
the bathing solutions, presumably because of buffering by the fixed negative charges on the surface of the lateral cell membranes.
To test the hypothesis that fixed buffers are responsible for the acidity, a theoretical and experimental approach was developed
for the determination of the concentration and pK of the fixed buffer constituted by the glycocalyx. The pH of the solution
in the LIS was measured by ratiometric fluorescence microscopy while the buffer concentration or composition of the bathing
solutions was altered. In addition, the divalent cation Sr2+ was added to the perfusion solutions to displace protons from the fixed buffer sites for the determination of the fixed buffer
properties. We conclude that the LIS contain 3.7 mm of pK 6.2 fixed buffer and that this buffer is responsible for the acidic microenvironment in the LIS.
Received: 9 April 1998/Revised: 28 July 1998 相似文献
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Igor Elman Tamara V. Gurvits Evelyne Tschibelu Justin D. Spring Natasha B. Lasko Roger K. Pitman 《PloS one》2013,8(4)
Increased neurological soft signs (NSSs) have been found in a number of neuropsychiatric syndromes, including chemical addiction. The present study examined NSSs related to perceptual-motor and visuospatial processing in a behavioral addiction viz., pathological gambling (PG). As compared to mentally healthy individuals, pathological gamblers displayed significantly poorer ability to copy two- and three-dimensional figures, to recognize objects against a background noise, and to orient in space on a road-map test. Results indicated that PG is associated with subtle cerebral cortical abnormalities. Further prospective clinical research is needed to address the NSSs'' origin and chronology (e.g., predate or follow the development of PG) as well as their response to therapeutic interventions and/or their ability to predict such a response. 相似文献
8.
[D-Ala2]Met-enkephalinamide (DALA) injected intracerebrally (IC) at low doses into specific sites of the mesencephalic reticular formation (MRF), produced a profound, long-lasting analgesia that was blocked by naloxone, a specific opiate antagonist. Morphine was only half as potent as DALA because morphine, injected IC at similar sites in the MRF, yielded a comparable analgesia only when injected at twice the dose. The analgesic effects of morphine were also antagonized by naloxene. Both DALA and morphine produced specific behavioral effects. Naloxone blocked the behavioral effects of DALA, but not those produced by morphine. 相似文献
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Summary The frequency of sister chromatid exchanges (SCEs) in the centromere of chromosomes involved in a whole-arm translocation t(1;19) was evaluated in altogether 911 metaphases of translocation carriers (n=5) and of normal controls (n=6). Comparison of the two groups reveals no significant differences in the SCE rate (x
2=3.06, n
f
=1). The question as to whether the possible increase of the SCE rate at the translocation point could be detected by light microscopy is discussed. Parameters included in the discussion are the ratio of the SCE frequency at the translocation point to the SCE frequency at any of the possible breakage points in the centromeric region and the number of possible breakage points in the centromeric region. 相似文献