Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

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.     

An automated technique for monitoring carbon dioxide respired by insects*

ABSTRACT. A non-dispersive infrared gas analyser equipped with a Luft-type sonic detector and flow-through reference cell was automated to monitor the total volume of carbon dioxide (CO₂) respired by single insects or groups of insects. The infrared analyser was interfaced with an integrator for quantification, a microprocessor to control intermittent air flow through the insect respiration chambers, and a microcomputer for data storage and reduction. This technique has been used to monitor the CO₂ Output of diapausing and non-diapausing mature fifth instar larvae and of developing pupae of the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). The resulting data were accurate, quantitative and reproducible.     

Subcellular distribution of enzymes in the yeast Saccharomycopsis lipolytica, grown on n-hexadecane,with special reference to the ω-hydroxylase

The subcellular localization of the ω-hydroxylase of Saccharomycopsis lipolytica was assessed by the analytical fractionation technique, originally described by de Duve C., Pressman, B.C., Gianetto, R., Wattiaux, R. and Appelmans, F., and hitherto little, if at all, applied to yeast. Protoplasts were separated in six fractions by differential centrifugation. Some of these fractions were further fractioned by density gradient centrifugation. The distribution of ω-hydroxylase and 15 other constituents chosen as possible markers of its subcellular membranes has been established. ω-Hydroxylase resulted in being bound to a membrane that containes also cytochrome P-450 and NADPH-cytochrome c reductase. This membrane clearly differs from five other subcellular entities. (1) Mitochondria were characterized by particulate malate dehydrogenase, particulate Antimycin A-insensitive NADH-cytochrome c reductase, oligomycin-sensitive and K⁺-stimulated ATPase pH 9. (2) Most if not all of the catalase and urate oxidase is peroxisomal. (3) Free ribosomes account for most RNA. (4) Nucleoside diphosphatase is for the first time reported in a yeast and appears to belong to an homogeneous population of small membranes. (5) The soluble compartment contains magnesium pyrophosphatase, alkaline phosphatase, 5′-nucleotidase and part of the NADH-cytochrome c reductase. Latent arylesterase and ATPase pH7 have an unspecific distribution. Alkaline phosphodiesterase I has not been detected.     

Retinal Attachment Instability Is Diversified among Mammalian Melanopsins

Melanopsins play a key role in non-visual photoreception in mammals. Their close phylogenetic relationship to the photopigments in invertebrate visual cells suggests they have evolved to acquire molecular characteristics that are more suited for their non-visual functions. Here we set out to identify such characteristics by comparing the molecular properties of mammalian melanopsin to those of invertebrate melanopsin and visual pigment. Our data show that the Schiff base linking the chromophore retinal to the protein is more susceptive to spontaneous cleavage in mammalian melanopsins. We also find this stability is highly diversified between mammalian species, being particularly unstable for human melanopsin. Through mutagenesis analyses, we find that this diversified stability is mainly due to parallel amino acid substitutions in extracellular regions. We propose that the different stability of the retinal attachment in melanopsins may contribute to functional tuning of non-visual photoreception in mammals.     

Binding of B‐cell maturation antigen to B‐cell activating factor induces survival of multiple myeloma cells by activating Akt and JNK signaling pathways

B‐cell maturation antigen (BCMA) is expressed on normal and malignant plasma cells and represents a potential target for therapeutic intervention. In this study, we characterized the mechanism underlying the protein kinase B (Akt) and c‐Jun N‐terminal kinase (JNK) pathways and BCMA interactions in regulating multiple myeloma (MM) cell survival. It was found that the expression levels of B cell‐activating factor (BAFF) and BCMA were increased in MM cells as compared with those in normal controls. The proliferation of U266 cells was induced by recombinant human BAFF (rhBAFF) and could also be decreased by BCMA siRNA. The expression of Bcl‐2 protein was up‐regulated, and Bax protein was down‐regulated after rhBAFF treatment, which could be reversed by BCMA siRNA. Similarly, the protein p‐JNK and p‐Akt were activated by rhBAFF and could be changed by BCMA siRNA. In addition, the BCMA mRNA and protein expression levels were decreased after treatment with Akt and JNK pathway inhibitors. These results suggest that Akt and JNK pathways are involved in the regulation of BCMA. A novel BAFF/BCMA signalling pathway in MM may be a new therapeutic target for MM. Copyright © 2016 John Wiley & Sons, Ltd.     

Pollen size-number trade-off and pollen-pistil relationships in Pedicularis (Orobanchaceae)

Current patterns of floral design in Pedicularis must have undergone an evolutionary process of interacting among components of floral traits, and then formed internal relationships among these traits. To detect such correlations, which may provide insight to understand flower evolution, 40 Pedicularis species representing all corolla types of the genus were studied. Results show that, interspecifically, pollen size correlates negatively with pollen number, but positively with pistil length. This suggests that plants evolve an optimal pollen size, which balances the advantages of large pollen size for gametophytic competition against the fecundity disadvantages of fewer pollen grains. In contrast to sex allocation theory, this study does not find a trade-off, but an interspecific positive correlation between pollen and ovule number. This is consistent with the hypothesis that genetic variation for resource acquisition may in part be responsible for the lack of negative correlation between male and female function.This work was supported by the State Key Basic Research and Development Plan, China (Grant No. G2000046804) to YHG. The authors would like to thank Peter K. Endress and two anonymous reviewers for providing critical comments and helpful suggestions, Qing-Feng Wang, Jing-Yuan Wang and Jin-Ming Chen for their helpful suggestions. Shi-Guo Sun, Jing Xia, and Qian Yu are thanked for their assistance in both the field work and laboratory phases of the project.     

The Administration of an α-MSH Analogue Reduces the Serum Release of IL-1α and TNFα Induced by the Injection of a Sublethal Dose of Lipopolysaccharides in the BALB/c Mouse

The injection of α-MSH or of one of its analogues ([Nle₄-D.Phe₇] α-MSH_4–10) reduced, in vivo, the release of two cytokines (IL-1α and TNFα) involved in inflammation. The inflammatory state was induced in BALB/c mice by intraperitoneal injection of a sublethal dose of lipopolysaccharides (LPS). The assay of these cytokines by ELISA showed a reduction of 20% with α-MSH and between 30 and 60% with the α-MSH analogue. The α-MSH or the analogue was administered in one of two ways: intravenously or subcutaneously. The most efficient method seemed to be the subcutaneous one because it improved the activity 10,000 times more than the intravenous method. Moreover, the analogue induced a regression of mortality in the animals treated by the intravenous method. Our results show that α-MSH and one of its analogues inhibit IL-1α and TNFα, and can be used as anti-inflammatory molecules.     

A tracer method to study unidirectional fluxes of lithium: Application to frog skin

We describe a new tracer method to measure unidirectional fluxes of Li⁺, despite the lack of any utilizable radioisotope of lithium. This method uses the purified stable isotopes, ⁶Li and ⁷Li, detected with an ion-probe microanalyser. The accuracy is comparable to that obtained for other ions (e.g., Na⁺) with radiotracers.The method has been applied to frog skin with both faces bathed in a 20% lithium/80% sodium medium. Sodium and lithium unidirectional fluxes have been measured simultaneously. The results are consistent with lithium being actively pumped, the outflux of lithium being, however, much larger than that of sodium.