•The interaction between MtRpsACTD and three specific RNA fragments were explored.•The RNA binding sites on MtRpsACTD locate in the S1 domain, comprised with the conserved residues.•Poly(A)-like sequence cooperates with sMLD mediating the MtRpsA-tmRNA interaction.•The charge and hydrogen-bonding interaction are the mainly interaction elements.•Our study offers a deep insight of the RpsA biochemical function in trans-translation.Ribosomal protein S1 (RpsA), the largest 30S protein in ribosome, plays a significant role in translation and trans-translation. In Mycobacterium tuberculosis, the C-terminus of RpsA is known as tuberculosis drug target of pyrazinoic acid, which inhibits the interaction between MtRpsA and tmRNA in trans-translation. However, the molecular mechanism underlying the interaction of MtRpsA with tmRNA remains unknown. We herein analyzed the interaction of the C-terminal domain of MtRpsA with three RNA fragments poly(A), sMLD and pre-sMLD. NMR titration analysis revealed that the RNA binding sites on MtRpsACTD are mainly located in the β2, β3 and β5 strands and the adjacent L3 loop of the S1 domain. Fluorescence experiments determined the MtRpsACTD binding to RNAs are in the micromolar affinity range. Sequence analysis also revealed conserved residues in the mapped RNA binding region. Residues L304, V305, G308, F310, H322, I323, R357 and I358 were verified to be the key residues influencing the interaction between MtRpsACTD and pre-sMLD. Molecular docking further confirmed that the poly(A)-like sequence and sMLD of tmRNA are all involved in the protein-RNA interaction, through charged interaction and hydrogen bonds. The results will be beneficial for designing new anti-tuberculosis drugs.

To investigate the effects of moxibustion on the serum metabolism in healthy human body based on the 1H nuclear magnetic resonance (1H NMR) metabolomics technology, and to find the differences in metabolites, as well as to elucidate the effects of moxibustion on healthy human body from the viewpoint of global metabolism.Sixty subjects of healthy young men from the enrolled students were randomly divided into a moxibustion group and a control group using random number table, with 30 cases in each group. Subjects in the moxibustion group accepted mild moxibustion on the right Zusanli (ST 36), once a day, 15 min for each time, and continuous treatment for 10 d; those in the control group did not receive any intervention. There were 28 cases in the moxibustion group and 23 cases in the control group after interventions. On the 1st day, 5th day and 10th day of the intervention, serum samples were collected from subjects of the two groups, and metabolic spectra were obtained by the 1H NMR technology.Before and after the intervention, serum 1H NMR of the moxibustion group was significantly different, while the difference was insignificant in the control group. Metabolite changes in the moxibustion group were mainly in low density lipoprotein (LDL)/very low density lipoprotein (VLDL), valine, isoleucine, leucine, lactic acid, glutamine, citric acid, polyunsaturated fatty acids, creatine, glycine, glycerol, glucose, tyrosine, histidine, formic acid, alanine, lysine, acetic acid, and glutamic acid.Moxibustion can cause changes of serum metabolic patterns in healthy human by influencing the concentrations of branched-chain amino acids, polyunsaturated fatty acids, and other metabolites to strengthen body's metabolisms of amino acids and fatty acid.基于核磁共振氢谱代谢组学技术(1H nuclear magnetic resonance, 1H NMR)探讨艾灸对正常人体血清代谢的影响, 并寻找差异性代谢物, 从整体代谢的角度阐述艾灸对健康人体的影响。将60 例在校健康青年男性采用随机数字表随机分成艾灸组和对照组, 每组30 例。艾灸组予温和灸右侧足三里, 每天1 次, 每次15 min, 连续治疗10 d; 对照组不予任何干预。干预结束后艾灸组剩余28 例, 对照组剩余23 例。在干预第1 d、第5 d 和第10 d, 采集两组受试者的血清样品, 运用1H NMR 技术获取代谢图谱。艾灸组干预前后血清1H NMR 有明显差异, 对照组干预前后1H NMR 无明显差异。艾灸组代谢物的变化主要是低密度脂蛋白(low densitylipoprotein, LDL)/极低密度脂蛋白(very low density lipoprotein, VLDL)、缬氨酸、异亮氨酸、亮氨酸、乳酸、谷氨酰胺、柠檬酸、多不饱和脂肪酸、肌酸、甘氨酸、甘油、葡萄糖、酪氨酸、组氨酸、甲酸、丙氨酸、赖氨酸、乙酸、谷氨酸。艾灸能够引起正常人体血清代谢模式变化, 通过影响支链氨基酸、多不饱和脂肪酸等代谢物浓度加强机体的氨基酸、脂肪酸代谢。

Cell culture metabolomics has demonstrated significant advantages in cancer research. However, its applications have been impeded by some influencing factors such as culture media, which could significantly affect cellular metabolic profiles and lead to inaccuracy and unreliability of comparative metabolomic analysis of cells. To evaluate the effects of different culture media on cellular metabolic profiling, we performed NMR-based metabolomic analysis of the human gastric cancer cell line SGC7901 cultured in both RPMI1640 and DMEM. We found that SGC7901 cultured in the two media exhibited distinct metabolic profiles with obviously different levels of discrepant metabolites, even though they showed almost the same cellular morphology and proliferation rates. When SGC7901 originally cultured in RPMI1640 was gradually acclimated in DMEM, both the metabolic profiles and most of the discrepant metabolite levels gradually converged toward those of the cells originally cultured in DMEM without significantly altered cell proliferation rates. However, several metabolite levels did not show the converging trends. Our results indicate that the effects of culture media on metabolic profiling must be carefully taken into account for comparative metabolomic analysis of cell lines. This work may be of benefit to the development of cell culture metabolomics.

Antimicrobial peptides (AMPs) play pivotal roles in the innate defense of vertebrates. A novel AMP (cathelicidin-PY) has been identified from the skin secretions of the frog Paa yunnanensis. Cathelicidin-PY has an amino acid sequence of RKCNFLCKLKEKLRTVITSHIDKVLRPQG. Nuclear magnetic resonance (NMR) spectroscopy analysis revealed that cathelicidin-PY adopts a tertiary structure with a mostly positively charged surface containing a helix (Thr15–Ser19). It possesses strong antimicrobial activity, low hemolytic activity, low cytotoxicity against RAW 264.7 cells, and strong anti-inflammatory activity. The action of antimicrobial activity of cathelicidin-PY is through the destruction of the cell membrane. Moreover, cathelicidin-PY exerts anti-inflammatory activity by inhibiting the production of nitric oxide (NO) and inflammatory cytokines such as tumor necrosis factor (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). Cathelicidin-PY inhibits the activation of Toll-like receptor 4 (TLR4) inflammatory response pathways induced by lipopolysaccharide (LPS). The NMR titration experiments indicated that cathelicidin-PY can bind to LPS. In conclusion, we have identified a novel potent peptide antibiotic with both antimicrobial and anti-inflammatory activities and laid the groundwork for future research and development.

β-Defensins are cationic, antimicrobial peptides that participate in antimicrobial defense as well as the regulation of innate and adaptive immunity. Human β-defensin 126 (DEFB126) is a multifunctional glycoprotein consisting of a conserved β-defensin core and a unique long glycosylated peptide tail. The long glycosylated peptide tail has been proven to be critical for efficient transport of sperm in the female reproductive tract, preventing their immune recognition, and efficient delivery of capacitated sperm to the site of fertilization. However, the functions of the conserved β-defensin core remain to be fully elucidated. In the present work, the conserved β-defensin core of the DEFB126 was expressed to explore its potential antimicrobial and anti-inflammatory activities. The DEFB126 core peptide exhibited both high potency for binding and neutralizing lipopolysaccharide (LPS) in vitro, and potent anti-inflammatory ability by down-regulating the mRNA expression of pro-inflammatory cytokines including IL-α, IL-1β, IL-6 and TNF-α in a murine macrophage cell line RAW264.7. The treatment with the DEFB126 core peptide also led to correspondingly decreased secretion of IL-6 and TNF-α. The blockade of LPS-induced p42/44 and p38 MAPK signal pathway might contribute to the anti-inflammation effects of the DEFB126 core peptide. Furthermore, fluorescence-labeled DEFB126 could enter RAW 264.7 cells and reduce the production of LPS-stimulated inflammatory factors, implying that DEFB126 might also participate in intracellular regulation beyond its direct LPS neutralization. In summary, our results demonstrate that the DEFB 126 core peptide has critical functions in parallel to its C-terminal tail by showing LPS-binding activity, anti-inflammatory effects and intracellular regulatory function.

Lipocalin 11 (Lcn11), a recently identified member of the lipocalin family, potentially plays crucial physiological roles in male reproduction. In this present work, we cloned, expressed and purified the rat Lcn11 (rLcn11) protein in Escherichia coli. A C59A/C156A substitution was introduced to ameliorate the misfolding and aggregation problem associated with the wild-type protein. From circular dichroism and non-reducing SDS–PAGE, we characterized the conformational properties of rLcn11 as a typical lipocalin scaffold with the conserved disulfide bridge. The results obtained from size-exclusion chromatography, cross-linking experiment and dynamic light scattering analysis indicate that the recombinant rLcn11 protein forms dimer in neutral solution. By using fluorescent probe 8-anilino-1-naphtahlene sulfonic acid (ANS), we found rLcn11 might contain multiple hydrophobic binding sites for ligand binding. Similarly to the odorant-binding protein, rLcn11 processes a moderate affinity for binding 1-aminoanthracene (AMA), implying that Lcn11 might work as a dimeric chemoreception protein in male reproductive system.Highlights► An efficient approach to express and purify rLcn11 protein and C59A/C156A mutant. ► The first study of the physicochemical properties of rLcn11 protein. ► The recombinant rLcn11 protein forms dimer in neutral solution. ► The C59A/C156A rLcn11 processes multiple binding sites for hydrophobic ligand. ► A moderate binding affinity of C59A/C156A rLcn11 for binding ligand AMA.

Cathelicidin Pc-CATH1 is a cathelicidin-derived myeloid antimicrobial peptide identified from Phasianus colchicus with strong antimicrobial activity against most of bacteria and fungi tested, including the clinically isolated (IS) drug-resistant strains. Considering the uniform distribution of net positive charge in both C- and N-terminus sequence of cathelicidin Pc-CATH1 and most of hydrophobic amino acid (aa) residues positioned in middle of the sequence, the antimicrobial peptide was used to investigate the structure-function relationship by truncating gradually N- or C-terminus amino acid residue. More than 10 modified peptide homologues (20–26 aa length) of cathelicidin Pc-CATH1 were found to keep strong antimicrobial abilities. The possible relationships between bioactivities including antimicrobial and hemolytic abilities, components of secondary structure, hydrophobicity, amphipathicity, net charge, and sequence length were investigated. The current work provided suggestions for structural and functional modification of linear, α-helical antimicrobial peptides containing no disulfided bridges. Open image in new window

A novel conotoxin, qc16a, was identified from the venom of vermivorous Conus quercinus. qc16a has only 11 amino acid residues, DCQPCGHNVCC, with a unique cysteine pattern. Its disulfide connectivity was determined to be I–IV, II–III. The NMR structure shows that qc16a adopts a ribbon conformation with a simple beta-turn motif formed by residues Gly6, His7 and Asn8. qc16a causes depression symptom in mice when injected intracranially. Point mutation results showed that Asp1, His7 and Asn8 are all essential for the activity of qc16a. Electrophysiologically, qc16a has no strong effect on the whole-cell currents of neurons and the currents of Drosophila Shaker channels, human BK channels and NaV1.7 channels. Its specific target still remains to be identified.Highlights► qc16a is a novel conotoxin with a rare C–C–CC framework. ► qc16a adopts a ribbon conformation with I–IV, II–III disulfide connectivity. ► Asp1, His7 and Asn8 are all essential for the activity of qc16a.

•The MtEspL protein with a purity of 98% and a yield of 14 mg/L was obtained.•1H-15N HSQC spectrum indicated MtEspL adopted a well-folded structure.•MtEspL exists in a dimeric form and possesses a DNA-binding activity.The Esx-1 cluster encodes a special secretion system that is important for granuloma formation and virulence when Mycobacterium tuberculosis infects the host. As one of the ‘core’ genes in the cluster, Rv3880c gene codes an Esx-1 secretion-associated protein EspL from Mycobacterium tuberculosis (MtEspL). It has been reported that EspL had a strong influence on the secretion of other two virulence factors, EsxA and EspE. However, so far little is known about the tertiary structure and specific function of MtEspL due to the difficulty in preparing the high-quality protein. In this study, we tried several fusion tags and various expression conditions to recombinantly express MtEspL. Through a four-step purification procedure, ultimately, we successfully prepared the full-length MtEspL in Escherichia coli BL21 (DE3) with a purity of 98%. The yields of the purified MtEspL protein were 14 mg/L in Luria Bertani medium and 5.6 mg/L in M9 minimal medium, respectively. Biophysical experiments showed that MtEspL existed in a dimeric form. Moreover, the 1H-15N HSQC spectrum recorded on MtEspL illustrates a favorable dispersion of the resonance peaks, indicating that the symmetric dimeric MtEspL adopted a well-folded structure and might be feasible to determine its solution structure by NMR spectroscopy. Moreover, we identified a strong DNA-binding ability of MtEspL with fluorescence quenching experiments. Our work lays the basis for further structural determination and functional exploration of MtEspL.

ObjectiveTo investigate the effect of fusion proteins expressed by the fused gene of porcine α 1, 3 galactosyltransferase (α1, 3 GT) and enhanced green fluorescent protein (EGFP) on the green fluorescence intensity of EGFP.MethodsThe fragment containing α 1, 3GT was firstly recovered after the pcDNA3.1- α 1, 3GT recombinant vector were digested with Bam HI and Eco RI, and then, the resultant fragment was ligated to the pEGFP-N1 vector which was also digested with the same enzymes. The new recombinant eukaryotic expression pEGFP/α 1, 3GT vector was obtained and sequenced. The pEGFP/α 1, 3GT was used to transfect human lung carcinoma cells A549 and HEKC 293FT, and the expression of EGFP was quantitatively analyzed by fluorescent microscope and flow cytometry.ResultsThe positive percentage of A549 was 80.5%, and that of 293 FT was 86.5% 48 hours after the two cell lines both were transfected by pEGFP-N1. The positive percentage of A549 was 75.8%, and that of 293 FT was 81.2% 48 hours after the two cell lines were transfected by pEGFP/α 1, 3GT. The mean fluorescence intensities of A549 transfected with pEGFP-N1 and pEGFP/α 1, 3GT were 1.21 and 0.956, respectively when compared with that of A549 without transfection. Meanwhile, the those of the 293FT that were transfected with pEGFP-N1 and pEGFP/α 1, 3GT were 7.66 and 1.00, respectively when compared with that of 293FT cells without transfection.ConclusionsThese results suggested that the expression of EGFP gene fused with porcine α 1, 3GT gene was partly inhibited.