Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.     

Effect of a hot water-soluble extraction from Grifola frondosa on the viability of a human monocyte cell line exposed to mitomycin C

The effects of a hot water-soluble extract of Grifola frondosa on the viability of multiple immune cell lines were examined in vitro. The extract and the 1000-cut extract, a low molecular weight fraction, improved the viability of a human monocyte cell line, THP-1, treated by the anticancer drug mitomycin C. DNA microarray and immunoassay results revealed increases in interleukin (IL)-8 expression by THP-1 cells in response to the 1000-cut extract. These data suggest that the extract would enhance the immune system through induction of IL-8 production by human blood monocytes, which leads to activated neutrophils.     

Aqueous extract of black tea (Camellia sinensis) prevents ethanol+cholecystokinin-induced pancreatitis in a rat model

Black Tea Extract (BTE), a phytocompound has been attributed with a plethora of health-promoting actions. We have previously demonstrated that BTE inhibits chronic hepatitis in a rat model induced with high-fat and ethanol (EtOH). This study reports that BTE prevents altered pancreatic acinar cell functions, oxidative stress, inflammatory changes and DNA damage in the EtOH+cholecystokinin (CCK)-induced model of pancreatitis. The EtOH+CCK model rats were administered with BTE, and were examined the activity of pancreatic digestive enzymes (amylase and lipase), proinflammatory cytokines (IL-6 and TNF-alpha), oxidative and antioxidative enzymes (nitric oxide, NO; malondialdehyde, MDA; superoxide dismutase, SOD; catalase, CAT), antioxidant level (glutathione, GSH), histopathological changes and the integrity of genomic DNA. Results show that because of chronic EtOH treatment, serum level of amylase and lipase (two biomarkers for pancreatitis) and pancreatic levels of MDA and NO (two biomarkers of oxidative stress) increased significantly, which could be effectively blunted by BTE. BTE could normalize EtOH+CCK-induced suppressed activities of SOD and CAT, and GSH content of pancreatic tissue. Also, histopathological and inflammatory changes during EtOH+CCK-induced pancreatitis could be blunted by BTE. Furthermore, BTE could effectively reduce EtOH+CCK-induced increase in DNA fragmentation and damage. These findings suggest that BTE prevents pancreatitis caused by chronic EtOH+CCK toxicity presumably by enhancing antioxidant, anti-inflammatory and antiapoptotic activity in rats.     

白萝卜提取物对病毒感染的保护作用

我们曾报道,葫芦科的丝瓜等食用植物组织的提取物有明显的抗病毒感染作用,并证明这些提取物为干扰素诱生剂。为了解其它种类植物是否也含有类似的物质,我们从十字花科植物——白萝卜(Raphanus sativus,white color,long form)成功地提取到类似物质。     

Target and non-target effects of Foeniculum vulgare and Matricaria chamomilla combined extract on Culex pipiens mosquitoes

The present study focused on extracting green larvicides from extracts of the combination of Foeniculum vulgare and Matricaria chamomilla using different solvents of increasing polarity in a Soxhlet extractor and evaluating their ovicidal, larvicidal, and cytotoxic activities. The most promising among all tested extracts was hexane extract. The ovicidal activity of the hexane PH2 extract resulted in a significant (p < 0.05) decrease in egg hatchability from 95.00 ± 6.16% to 15 ± 9.04% at doses ranging from 62.5 to 500 µg/mL. The larval mortality with the hexane extract ranged from 13.33 ± 3.3% to 93.33 ± 3.3% at doses ranging from 31.25 to 250 µg/mL, respectively. The LC₅₀ and LC₉₀ values of the larvicidal activity of the hexane extract were estimated to be 148.3 and 242.17 µg/mL, respectively, after 24 h of exposure. Similarly, the LC₅₀ values after 48 and 72 h of exposure were 124.93 and 100.3 µg/mL, respectively, against the third instar of Cx. pipiens. PH2 treatment of larvae resulted in histopathological changes such as degenerated epithelial cells and destruction of microvilli on the epithelial cells. The PH2 extract achieved a dose-dependent decrease in the rate of cell survival. The IC₅₀ value of PH2-treated HUVECs was 192.07 µg/mL after 24 h of incubation. The cells showed changes in cellular and nuclear morphology. In conclusion, the hexane extract of PH2 could be used in mosquito management programs.     

乌头须根总生物碱提取工艺的考察

目的:考察乌头须根中总生物碱的最佳提取工艺条件。方法:酸碱滴定法测定乌头须根中总生物碱含量,以总生物碱提取率为指标,采用L9(3)~4正交实验法筛选乌头须根总生物碱的最佳提取工艺。结果:乌头须根总生物碱含量为1.094%,影响提取的主次因素为:乙醇浓度>提取次数>提取时间>乙醇用量;优选得到的最佳提取工艺为A_3B_1C_3D_3,即以8倍量80%的乙醇提取3次,每次1.5小时。结论:乌头须根总生物碱含量较高,提取工艺条件稳定、经济、可行。     

Zinc supplementation decreases hepatic copper accumulation in LEC rat

The effect of dietary zinc (Zn) supplementation on copper (Cu)-induced liver damage was investigated in Long-Evans Cinnamon rats (LEC), a model for Wilson's disease (WD). Four-week-old LEC (N=64) and control Long-Evans (LE) (N=32) female rats were divided into two groups; one group was fed with a Zn-supplemented diet (group I) and the other was given a normal rodent diet (group II). LEC rats were killed at 6, 8, 10, 12, 18, and 20 wk of age; the LE control rats were killed at 6, 12, 18, and 20 wk of age. Cu concentration in the liver was reduced in LEC rats fed the Zn-supplemented diet compared with LEC rats on the normal diet between 6 and 18 wk of age. Metallothionein (MT) concentration in the livers of LEC rats in group I increased between 12 and 20 wk of age, whereas hepatic MT concentration in LEC rats from group II decreased after 12 wk. Hepatocyte apoptosis, as determined by TUNEL, was reduced in Zn-supplemented LEC rats at all ages. Cholangiocellular carcinoma was observed only in LEC rats in group II at wk 20. These results suggest that Zn supplementation can reduce hepatic Cu concentration and delay the onset of clinical and pathological changes of Cu toxicity in LEC rats. Although the actual mechanism of protection is unknown, it could be explained by sequestration of dietary Cu by intestinal MT, induced by high dietary Zn content.     

非嗜食植物提取物对桔全爪螨产卵的驱避性测定

The oviposition repellency of the alcohol extracts from 50 species of non-preferable plants and azadirachtin against citrus red mite ( Panonychus cirri ) was determined using laboratory bioassays. In choice tests, the extracts from 42 of the 50 plant species and 1% azadirachtin (2000×) significantly reduced oviposition 1d after treatment.The repellency effect of the extracts from the 42 plant species was better than that of azadirachtin. Mikania micrantha extract had the best result, with an Interference Index of Population Control (IIPC) of 0.087 1 d after treatment, and significantly reduced oviposition for 9 d, while the extracts from Sesbania cannabina, Allium tuberosum , Paederia scandens , Duranta erecta and Dicranopteris pedata also had good effects, with an oviposition repellency of over 70 % 1 d after treatment, and significantly reduced oviposition for 4 - 6 d. The effect became weaker as time went on. None of the extracts showed significant oviposition attraction.     

不同浓度亳菊水提物对秀丽隐杆线虫脂滴凝聚影响观察

目的探讨毫菊对秀丽隐杆线虫脂肪沉积的影响。方法用不同浓度毫菊水提取液配制NGM培养基,培养同步化秀丽隐杆线虫,成虫经油红0染色,异丙醇萃取,测定OD值,并与普通NGM培养基培养的同步化线虫相比较。结果随着浓度的提高,线虫体内的脂肪沉积量呈现出一定的曲线变化,其具体机制有待于进一步研究。     

Inactivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 by cinnamon extracts in carrot-kinnow mandarin blends

We investigated the antimicrobial efficacy of cinnamon extracts in laboratory prepared Kinnow-mandarin carrot blends challenged with Escerichia coli O157:H7. Freshly squeezed carrot and kinnow-mandarin juices were mixed to obtain a typical blend, inoculated with E. coli O157:H7 cultures at 10² CFU/mL with and without cinnamon extracts (0.3%) and stored at 4, 8 and 28°C for up to 10 hours. Counts on tryptic soy agar (TSA) selective medium (Mac conkey sorbitol agar) and thin agar layer (TAL) were determined at every 2 hour. The TAL method was used for recovery of sublethally injured cells. Inactivation of E. coli O157:H7 in blends containing 0.3% cinnamon extracts were observed, with killing effects being more pronounced at 28°C, similar trends were evident with blends stored under refrigeration conditions (4 and 8°C).The decrease in counts were attributed to several factors namely, pH, storage temperature and addition of cinnamon. The results of our study indicate that cinnamon extracts could be used as an effective, natural antimicrobial for assuring consumer safety at the point of preparation of carrot-kinnow mandarin blends, which is a popular, nutritional beverage consumed in India.