RESUMEN
Nitrile imines produced by photodissociation of 2,5-diaryltetrazoles undergo cross-linking reactions with amide groups in peptide-tetrazole (tet-peptide) conjugates and a tet-peptide-dinucleotide complex. Tetrazole photodissociation in gas-phase ions is efficient, achieving ca. 50% conversion with 2 laser pulses at 250 nm. The formation of cross-links was detected by CID-MS3 that showed structure-significant dissociations by loss of side-chain groups and internal peptide segments. The structure and composition of cross-linking products were established by a combination of UV-vis action spectroscopy and cyclic ion mobility mass spectrometry (c-IMS). The experimental absorption bands were found to match the bands calculated for vibronic absorption spectra of nitrile imines and cross-linked hydrazone isomers. The calculated collision cross sections (CCSth) for these ions were related to the matching experimental CCSexp from multipass c-IMS measurements. Loss of N2 from tet-peptide conjugates was calculated to be a mildly endothermic reaction with ΔH0 = 80 kJ mol-1 in the gas phase. The excess energy in the photolytically formed nitrile imine is thought to drive endothermic proton transfer, followed by exothermic cyclization to a sterically accessible peptide amide group. The exothermic nitrile imine reaction with peptide amides is promoted by proton transfer and may involve an initial [3 + 2] cycloaddition followed by cleavage of the oxadiazole intermediate. Nucleophilic groups, such as cysteine thiol, did not compete with the amide cyclization. Nitrile imine cross-linking to 2'-deoxycytidylguanosine was found to be >80% efficient and highly specific in targeting guanine. The further potential for exploring nitrile-imine cross-linking for biomolecular structure analysis is discussed.
Asunto(s)
Iminas , Protones , Iminas/química , Nitrilos , Péptidos/química , Iones , Amidas/química , Oligonucleótidos , Tetrazoles/químicaRESUMEN
d(GCGAAGC) is the smallest oligonucleotide with a well-defined hairpin structure in solution. We report a study of multiply protonated d(GCGAAGC) and its sequence-scrambled isomers, d(CGAAGCG), d(GCGAACG), and d(CGGAAGC), that were produced by electrospray ionization with the goal of investigating their gas-phase structures and dissociations. Cyclic ion mobility measurements revealed that dications of d(GCGAAGC) as well as the scrambled-sequence ions were mixtures of protomers and/or conformers that had collision cross sections (CCS) within a 439-481 Å2 range. Multiple ion conformers were obtained by electrospray under native conditions as well as from aqueous methanol. Arrival time distribution profiles were characteristic of individual isomeric heptanucleotides. Extensive Born-Oppenheimer molecular dynamics (BOMD) and density functional theory (DFT) calculations of d(GCGAAGC)2+ isomers indicated that hairpin structures were high-energy isomers of more compact distorted conformers. Protonation caused a break up of the C2···G6 pair that was associated with the formation of strong hydrogen bonds in zwitterionic phosphate anion-nucleobase cation motifs that predominated in low energy ions. Multiple components were also obtained for d(GCGAAGC)3+ trications under native and denaturing electrospray conditions. The calculated trication structures showed disruption of the G···C pairs in low energy zwitterions. A hairpin trication was calculated to be a high energy isomer. d(GCGAAGC)4+ tetracations were produced and separated by c-IMS as two major isomers. All low energy d(GCGAAGC)4+ ions obtained by DFT geometry optimizations were zwitterions in which all five purine bases were protonated, and the ion charge was balanced by a phosphate anion. Tetracations of the scrambled sequences were each formed as one dominant isomer. The CCS calculated with the MobCal-MPI method were found to closely match experimental values. Collision-induced dissociation (CID) spectra of multiply charged heptanucleotides showed nucleobase loss and backbone cleavages occurring chiefly at the terminal nucleosides. Electron-transfer-CID tandem mass spectra were used to investigate dissociations of different charge and spin states of charge-reduced heptanucleotide cation radicals.
RESUMEN
Royal jelly (RJ) from honeybee has been widely used as a health promotion supplement. The major royal jelly proteins (MRJPs) have been identified as the functional component of RJ. However, the question of whether MRJPs have anti-senescence activity for human cells remains. Human embryonic lung fibroblast (HFL-I) cells were cultured in media containing no MRJPs (A), MRJPs at 0.1 mg/ml (B), 0.2 mg/ml (C), or 0.3 mg/ml (D), or bovine serum albumin (BSA) at 0.2 mg/ml (E). The mean population doubling levels of cells in media B, C, D, and E were increased by 12.4%, 31.2%, 24.0%, and 10.4%, respectively, compared with that in medium A. The cells in medium C also exhibited the highest relative proliferation activity, the lowest senescence, and the longest telomeres. Moreover, MRJPs up-regulated the expression of superoxide dismutase-1 (SOD1) and down-regulated the expression of mammalian target of rapamycin (MTOR), catenin beta like-1 (CTNNB1), and tumor protein p53 (TP53). Raman spectra analysis showed that there were two unique bands related to DNA synthesis materials, amide carbonyl group vibrations and aromatic hydrogens. These results suggest that MRJPs possess anti-senescence activity for the HFL-I cell line, and provide new knowledge illustrating the molecular mechanism of MRJPs as anti-senescence factors.
Asunto(s)
Senescencia Celular/efectos de los fármacos , Ácidos Grasos/química , Fibroblastos/citología , Pulmón/citología , Animales , Abejas , Bovinos , Línea Celular , Proliferación Celular , Medios de Cultivo , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Humanos , Proteínas de Insectos/química , Pulmón/efectos de los fármacos , Albúmina Sérica/metabolismo , Espectrometría Raman , Superóxido Dismutasa-1/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , beta Catenina/metabolismoRESUMEN
Turmeric residue (TR), containing residual levels of curcumin, is a solid by-product waste generated after the extraction and separation of curcumin from turmeric root. A feeding trial was conducted to evaluate the effects of TR on the survival of Chinese soft-shelled turtles (SSTs), Pelodiscus sinensis, under a high ambient temperature. A total of 320 female SSTs were assigned randomly to two diets: basal diet (the control group, n=160) and an interventional diet supplemented with 10% TR (the TR group, n=160). Our results demonstrated that supplementation of TR increased the SST survival rate by 135.5%, and superoxide dismutase (SOD) activity of SST liver by 112.8%, and decreased the malondialdehyde (MDA) content of SST liver by 36.4%, compared to the control group. The skin of the SST fed TR showed a golden color. High-performance liquid chromatography (HPLC) analysis indicated that the concentrations of curcumin in TR and the skin of the SST fed TR were (1.69±0.30) and (0.14±0.03) µg/g, respectively. Our observation suggests that supplementation of TR increased the survival rate of SST under high ambient temperatures. We speculated that the increased survival rate and tolerance at the high ambient temperature were associated with the anti-oxidation activity of curcumin from TR. Moreover, curcumin in TR could be deposited in SST skin, which made it more favored in the market of China. Our findings provide new knowledge and evidence to effectively reuse TR as a feed additive in animal and aquatic farming.
Asunto(s)
Alimentación Animal , Extractos Vegetales/farmacología , Tortugas/fisiología , Animales , Antioxidantes/farmacología , Peso Corporal/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Curcuma , Curcumina/análisis , Suplementos Dietéticos , Femenino , Calor , Extractos Vegetales/análisis , Superóxido Dismutasa/metabolismo , Tasa de SupervivenciaRESUMEN
Royal jelly (RJ) is a well-known bioactive substance. It contains large amounts of major royal jelly proteins (MRJPs), which express growth-factor-like activity in several animal and human cell lines. However, the question on whether MRJPs possess growth-factor-like activity on all types of cell cultures remains. In order to determine whether MRJPs can be used as an alternative to fetal bovine serum (FBS) in different types of human cell culture, the proliferation of the complex serum with different ratios of MRJPs/FBS (M/F) was evaluated on five cell lines: 293T, HFL-I, 231, HCT116, and Changliver using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. The proliferation activity of the combination of the complex M/F serum with cytokines on the test cell lines was also measured. The results demonstrated that the complex serum with M/F 6/4 possessed the highest proliferation activity similar to or in excess of FBS. However, no activity of complex medium with M/F 6/4 was observed in 231 cells, indicating a selectivity of MRJPs on cell types. Compared with the complex medium with M/F 6/4, the complex medium with M/F 6/4 together with two cytokines, epidermal growth factor (EGF) and insulin-transferrin-selenium (ITS), promoted proliferations of Changliver, 293T, HCT116, and HFL-I by 18.73%â56.19% (P<0.01). Our findings demonstrate that MRJPs could partially replace FBS in culturing many human cell lines.