[4]Helicene–Squalene Fluorescent Nanoassemblies for Specific Targeting of Mitochondria in Live‐Cell Imaging

Ester, amide, and directly linked composites of squalene and cationic diaza [4]helicenes 1 are readily prepared. These lipid‐dye constructs 2 , 3 , and 4 give in aqueous media monodispersed spherical nanoassemblies around 100–130 nm in diameter with excellent stability for several months. Racemic and enantiopure nanoassemblies of compound 2 are fully characterized, including by transmission electron microscope and cryogenic transmission electron microscope imaging that did not reveal higher order supramolecular structures. Investigations of their (chir)optical properties show red absorption maxima ≈600 nm and red fluorescence spanning up to the near‐infrared region, with average Stokes shifts of 1350–1550 cm^?1. Live‐cell imaging by confocal microscopy reveals rapid internalization on the minute time scale and organelle‐specific accumulation. Colocalization with MitoTracker in several cancer cell lines demonstrates a specific staining of mitochondria by the [4]helicene–squalene nanoassemblies. To our knowledge, it is the first report of a subcellular targeting by squalene‐based nanoassemblies.     

Engineering of Saccharomyces cerevisiae for the production of (+)-ambrein

The triterpenoid (+)-ambrein is the major component of ambergris, a coprolite of the sperm whale that can only be rarely found on shores. Upon oxidative degradation of (+)-ambrein, several fragrance molecules are formed, amongst them (−)-ambrox, one of the highest valued compounds in the perfume industry. In order to generate a Saccharomyces cerevisiae whole-cell biocatalyst for the production of (+)-ambrein, intracellular supply of the squalene was enhanced by overexpression of two central enzymes in the mevalonate and sterol biosynthesis pathway, namely the N-terminally truncated 3-hydroxy-3-methylglutaryl-CoA reductase 1 (tHMG) and the squalene synthase (ERG9). In addition, another key enzyme in sterol biosynthesis, squalene epoxidase (ERG1) was inhibited by an experimentally defined amount of the inhibitor terbinafine in order to reduce flux of squalene towards ergosterol biosynthesis while retaining sufficient activity to maintain cell viability and growth. Heterologous expression of a promiscuous variant of Bacillus megaterium tetraprenyl-β-curcumene cyclase (BmeTC-D373C), which has been shown to be able to catalyse the conversion of squalene to 3-deoxyachillol and then further to (+)-ambrein resulted in production of these triterpenoids in S. cerevisiae for the first time. Triterpenoid yields are comparable with the best microbial production chassis described in literature so far, the methylotrophic yeast Pichia pastoris. Consequently, we discuss similarities and differences of these two yeast species when applied for whole-cell (+)-ambrein production.     

高效液相色谱法测定角鲨烯软胶囊中的角鲨烯

目的建立高效液相色谱法测定角鲨烯软胶囊中角鲨烯的含量。方法样品经甲醇提取,以甲醇/乙腈(V:V=60:40)为流动相,流速为1.2 m L/min,经高效液相色谱在色谱柱Waters Spherisorb C18分离,柱温为40℃;然后在检测波长为203 nm的条件下测定角鲨烯的含量。结果角鲨烯的浓度在0.1023~0.5118 mg/m L的范围内与峰面积线性关系良好,相关系数r0.999,方法的检出限为0.0025%,在不同添加水平下,方法的平均回收率为100.5%,相对标准偏差为0.6%(n=9)。与我国《国家药品标准》中WS-1001-(HD-080-18)-2002角鲨烯气相色谱法测定比较,两种结果无显著差异。结论高效液相色谱法更快捷简单,节省检测成本,可应用于保健食品中角鲨烯的含量测定。     

Capillary supercritical fluid chromatographic analysis of shark liver oils

The liver oils of six different shark species have been analyzed by supercritical fluid chromatography (SFC). The liver oils were from the species Pseudotriakis microdon (False catshark), Centroscymnus coelepsis (Portuguese dogfish), Centrophorus squanosus (Leafscalp gulper shark), Deanea calceus (Birdbeak dogfish), Etmopterus princips (Greater lantern shark), and Centroscymnus crepidater (Longnose velvet dogfish). The method was capable of direct quantitation of squalene and cholesterol, while quantitation of triacylglycerols, cholesterol esters, and diacylglycerol ethers required thin-layer chromatographic fractionation prior to SFC analysis. The iodine values of the liver oil samples gave a linear correlation when plotted against the squalene content found by SFC. The variation of squalene content within one shark species is large, and there are large differences in squalene content from species to species. The squalene contents varied between 0.22 and 82.54 wt%. The identity of the glycerol ethers was investigated by SFC of the unsaponifiable matter. The major glycerol ethers contained chimyl, batyl, and selachyl alcohol.     

Associations between Oil Yield and Profile of Fatty Acids,Sterols, Squalene,Carotenoids, and Tocopherols in Seed Oil of Selected Japanese Quince Genotypes during Fruit Development

Tocopherols, phytosterols, carotenoids, and squalene are present in mature seeds of Japanese quince. Yet, little is known about the relationship between these compounds and oil yield during fruit and seed development. The profile change of lipophilic compounds during fruit and seed development in Japanese quince cultivars “Darius,” “Rondo,” and “Rasa” is investigated. It is shown here that during fruit and seed development, there is a significant reduction, three‐ to over tenfold, in the concentration of minor bioactive compounds in seed oil. It is recorded that delay between synthesis of tocopherols and oil in Japanese quince seeds during the fruit development results in a logarithmic relationship between the oil content and tocopherols concentration in the seed oil (R² = 0.980). Similar trends are observed between oil yield and phytosterols, and carotenoids (R² = 0.927 and R² = 0.959, respectively). The profile of fatty acids during the development of the seeds significantly is changed. The reduction of linoleic, palmitic, and gondoic acids levels and increment of oleic acid is noted. The oil content, profile of fatty acids, and concentration of bioactive compounds in all three genotypes of Japanese quince do not change significantly statistically during the last month of fruit development. Practical Applications: Some fruits are harvested at different degrees of maturity mainly due to a logistic issue and uneven ripening of fruits, which affects the chemical composition of whole fruit including seeds. Therefore, it would be good to know how the chemical composition is changing in plant material during development especially in the last month before harvest. Production of Japanese quince continues to rise year to year and with it the volume of generated by‐products such as seeds. This study demonstrates how it changes the oil content, profile of fatty acid, and concentration of tocopherols, squalene, phytosterols, and carotenoids in the seeds and seed oil of three Japanese quince cultivars “Rondo,” “Darius,” and “Rasa” during plant development. The provided information can be very useful for the manufactories oriented on the processing of by‐products, mainly seeds, generated by other branches of industry, for instance, fruit‐processing.     

Synthesis,Computational Studies and Assessment of in Vitro Activity of Squalene Derivatives as Carbonic Anhydrase Inhibitors

We report novel molecules incorporating the nontoxic squalene scaffold and different carbonic anhydrase inhibitors (CAIs). Potent inhibitory action, in the low-nanomolar range, was detected against isoforms hCA II for sulfonamide derivatives, which proved to be selective against this isoform over the tumor-associate hCA IX and XII isoforms. On the other hand, coumarin derivatives showed weak potency but high selectivity against the tumor-associated isoform CA IX. These compounds are interesting candidates for preclinical evaluation in glaucoma or various tumors in which the two enzymes are involved. In addition, an in silico study of inhibitor-bound hCA II revealed extensive interactions with the hydrophobic pocket of the active site and provided molecular insights into the binding properties of these new inhibitors.     

A Novel Function of the Triterpene Squalene in a Tritrophic System

Changes in apple leaf chemistry after infestation by leafminers and their effect on both host location and host habitat location of the generalist parasitoid Pholetesor bicolor were investigated. Chemical analysis of leaf solvent extracts from healthy and leafminer-damaged leaves revealed that herbivory increased the amount of the triterpene squalene (C₃₀H₅₀), whereas quantities of all other identified compounds were similar in both plant treatments. To assess the response of parasitoids to host location cues, contact bioassays were conducted with naïve females. Results showed that parasitoids performed a characteristic ovipositional probing more often on the mine-damaged than on the healthy leaf. This behavior was triggered by a hexane extract of the mine-damaged leaf, but not by a healthy leaf extract. A synthetic mixture of the compounds identified in the extract triggered a similar response. A mixture devoid of squalene was not active, whereas squalene alone elicited the probing behavior. To assess the use of the identified compounds in habitat location, Y-tube olfactometer experiments were conducted with naïve and experienced females. Results showed that squalene is not involved in habitat location and has no priming effect on P. bicolor. While other triterpenes are known to mediate habitat location of parasitoids, this is the first report in which a plant triterpene is shown to mediate host location of a parasitoid. The biological and ecological functions of squalene on all three trophic levels are discussed.     

Head‐to‐Head Prenyl Synthases in Pathogenic Bacteria

Many organisms contain head‐to‐head isoprenoid synthases; we investigated three such types of enzymes from the pathogens Neisseria meningitidis, Neisseria gonorrhoeae, and Enterococcus hirae. The E. hirae enzyme was found to produce dehydrosqualene, and we solved an inhibitor‐bound structure that revealed a fold similar to that of CrtM from Staphylococcus aureus. In contrast, the homologous proteins from Neisseria spp. carried out only the first half of the reaction, yielding presqualene diphosphate (PSPP). Based on product analyses, bioinformatics, and mutagenesis, we concluded that the Neisseria proteins were HpnDs (PSPP synthases). The differences in chemical reactivity to CrtM were due, at least in part, to the presence of a PSPP‐stabilizing arginine in the HpnDs, decreasing the rate of dehydrosqualene biosynthesis. These results show that not only S. aureus but also other bacterial pathogens contain head‐to‐head prenyl synthases, although their biological functions remain to be elucidated.     

不同瓜类种子性状及其油中角鲨烯含量的分析比较

为探究不同瓜类种子性状与含油量及其油中角鲨烯含量的相关性,以苦瓜、瓜蒌、籽瓜和南瓜等15个品种的瓜类种子为实验材料,对单粒质量、百粒质量、长、宽、干基含油量和油中角鲨烯含量进行测定,并分析其相关性。结果表明：籽瓜种子单粒质量最大、长度最长、宽度最宽,分别为0.255 2 g、0.632 mm和0.383 mm;阿根廷蛇瓜种子百粒质量最大,为24.919 3 g;砍瓜种子的干基含油量和油中角鲨烯含量最高,分别为41.14%和2 525.8 mg/kg;相关性分析发现种子长度越长,宽度越宽有利于单粒质量和百粒质量的增加,油中角鲨烯含量与干基含油量呈显著正相关(p<0.05);西葫芦、青皮绿肉酥、砍瓜、早熟南瓜和南瓜在籽油角鲨烯含量选育中具有一定的参考价值。综上,砍瓜为种子高含油量及其油中高角鲨烯含量的瓜类品种。