Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins. The current methods that applied in protein-protein interaction, such as co-immunoprecipitation and pull down etc., often cause plenty of working time due to the burdensome cloning and purification procedures. Here we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within E. coli system. We modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease, two cleavage site F and two multiple cloning sites that flanking cleavage sites. The target proteins (for example: protein A and protein B) were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His(6) tag. PPV NIa protease carried out intracellular cleavage along expression, then led to the separation of polyprotein components, therefore, the interaction between protein A-protein B can be detected through one-step purification and analysis. Negative control for protein B was brought into this system for monitoring interaction specificity. We successfully employed this system to prove two cases of reported protien-protein interaction: RHA2a/ANAC and FTA/FTB. In conclusion, a convenient and efficient system has been successfully developed for detecting protein-protein interaction.
Endopeptidases ; genetics ; metabolism ; Escherichia coli ; genetics ; Plum Pox Virus ; enzymology ; genetics ; Protein Interaction Mapping ; methods ; Proteolysis

Background Long-term exposure to hand-transmitted vibration can lead to hand-arm vibration syndrome, one manifestation of which is impaired peripheral blood circulation in the arms. Altered expressions of prostacyclin I₂ (PGI₂) and thromboxane A₂ (TXA₂) in blood may be one of the important mechanisms of vibration-induced hand-arm vibration syndrome. Objective To reveal the effects of rat tail vibration on the expressions of PGI₂ and TXA₂ in plasma, and to establish the correlation between the change of rat plasma PGI₂ to TXA₂ ratio and rat tail vibration. Methods Fifty SPF-grade male SD rats were randomly divided into five groups: control group, 1 d exposure group, 3 d exposure group, 7 d exposure group, and 14 d exposure group, with 10 rats in each group. The rats were placed in rat immobilizes on a immobilization table, and the rats' tails were connected to a shaker and fixed with medical tape. There was no overlap between the immobilizes and between the rats' tails by no contact between the immobilization table and the shaker. The exposure dose was 125 Hz, 5.9 m·s⁻², 4 h·d⁻¹, and the vibration direction was linear vertical vibration. Abdominal aortic blood was taken at the end of vibration exposure, and the expressions of PGI₂, TXA₂, and their hydrolysates 6-keto-prostaglandin F_1α (6-keto-PGF_1α) and thromboxane B₂ (TXB₂) were measured by enzyme-linked immunosorbent assay, and the 6-keto-PGF_1α/TXB₂ values were calculated. Spearman rank correlation was used to analyze whether the expression of vascular factors correlated with the accumulated time of vibration. Results The expression levels of plasma 6-keto-PGF_1α were (896.12±124.37), (1068.13±119.41), (1215.26±122.64), and (1317.94±106.54) ng·L⁻¹ in the 1 d, 3 d, 7 d, and 14 d groups of rats, respectively, which were higher than that in the control group, (830.60±109.47) ng·L⁻¹ (P<0.001). The PGI₂ expression levels were (86.49±2.40), (107.90±2.65), (114.02±2.16), and (126.95±1.94) ng·L⁻¹ in the 1 d, 3 d, 7 d, and 14 d groups of rats, respectively, all higher than (60.09±2.11) ng·L⁻¹ in the control group (P＜0.001). The expression levels of TXB₂ were (132.14±4.10), (145.52±4.09), (179.91±4.98), and (204.10±3.22) ng·L⁻¹ in the 1 d, 3 d, 7 d, and 14 d groups of rats, respectively, which were higher than that in the control group, (106.08±3.26) ng·L⁻¹ (P<0.001). The expression levels of plasma TXA₂ were (211.99±3.24), (236.33±3.88), and (245.45±4.23) ng·L⁻¹ in rats in the 3 d, 7 d, and 14 d groups, respectively, which were all elevated compared with (174.79±4.19) ng·L⁻¹ in the control group (P<0.001). Compared with the control group, the 6-keto-PGF_1α/TXB₂ values were decreased in the 7 d and 14 d groups (P<0.05). The 6-keto-PGF_1α, PGI₂, TXB₂, and TXA₂ expressions were positively correlated with vibration accumulation time (r=0.84, 0.84, 0.80, 0.84, P<0.001) and the 6-keto-PGF_1α/TXB₂ values were negatively correlated with vibration accumulation time (r=-0.24, P=0.003). Conclusion Local exposure of rat tail to vibration could increase the expressions of PGI₂ and TXA₂ in blood, and the elevated expressions show a dose-effect relationship with the duration of vibration exposure, but the PGI₂/TXA₂ tends to decrease with the accumulation of vibration exposure.

Background The metabolites and metabolic pathways of hand-arm vibration syndrome have not yet been elucidated. Objective To investigate the effect of local vibration on endogenous metabolites in rat serum by metabolomic analysis, to preliminarily explore the potential metabolic pathway of endogenous metabolites, so as to provide evidence for further research on the mechanism of hand-arm vibration syndrome. Methods Thirty-two SPF male SD rats, (211.3±11.1) g, 7−8 weeks of age, were selected and randomly divided into three groups: control group (14 rats, without vibration), 7 d vibration group (9 rats, continuously vibration for 7 d), and 14 d vibration group (9 rats, continuous vibration for 14 d). The vibration rats were vibrated every day for 4 h, the frequency weighted acceleration was 4.9 m·s⁻², the vibration frequency was 125 Hz, and the vibration direction was one-way vertical vibration. The control group had the same conditions except not contacting vibration. After the vibration exposure, the blood samples taken from the abnormal aorta of rats were collected, and the changes of rat serum metabolome were analyzed by ultra-performance liquid chromatography-tandem time-of-flight mass spectrometry. Principal components analysis (PCA) was used to explore changes in rat serum metabolic profile, and orthogonal partial least squares-discriminant analysis (OPLS-DA) was used to screen out differential metabolites. Combined with online databases, a metabolic pathway enrichment analysis of differential metabolites was performed. Results The PCA analysis showed that compared with the control group, the rat serum metabolic profiles in the 7 d group and the 14 d group were clearly differentiated, and the rat serum metabolic profiles in the 7 d group and the 14 d group partially overlapped. The OPLS-DA analysis showed significant differences between groups. The main parameters were: model interpretation rate R²Y=0.914, model predictive ability Q²=0.58. The OPLS-DA analysis screened out 26 and 119 differential metabolites from the 7 d group and the 14 d group respectively, and there were 24 common differential metabolites between the 7 d group and the 14 d group. The metabolomic pathway analysis showed that local vibration-induced changes in rat serum metabolism were mainly related to arachidonic acid metabolism in the 14 d group, among which the metabolites with significant effects were arachidonic acid, prostaglandin E₂, and prostaglandin D₂. Conclusion Local vibration could affect the normal metabolism in rats, and the metabolic pathway with significant influence is arachidonic acid metabolism after a 14 d exposure and the involved metabolites are arachidonic acid, prostaglandin E₂, and prostaglandin D₂.

AIM: Lipopolysaccharide (LPS) on the cell membrane of gram negative bacteria is closely related to the occurrence and development of severe acute pancreatitis (SAP). Local and systemic monocyte / macrophages play an important role in the inflammatory process of SAP. Artesunate (AS) was reported to protect rats with severe acute pancreatitis by reducing the release of proinflammatory cytokines. This study further explored the molecular mechanism of anti-inflammatory effect of AS. METHODS: The release of proinflammatory cytokines in the supernatant were studied by enzyme-linked immunosorbent assay. Then, the mRNA expressions of PI3K-III and its key molecules in signaling pathway were detected by real-time quantitative PCR. Finally, the phosphorylation levels of PI3KIII were detected by Western blot. RESULTS: AS could significantly inhibit the release of proinflammatory cytokines from mouse macrophage induced by LPS. Autophagy inhibitor 3-methyladenine (3-MA) could significantly inhibit the release of TNF-α from mouse macrophages induced by LPS; LPS significantly increased the mRNA expression of PI3KIII and its key molecules in mouse peritoneal macrophages (PMs). Finally, AS could significantly inhibit the increase of PI3K-III phosphorylation induced by LPS in PMs. CONCLUSION: The anti-inflammatory mechanism of AS is closely related to the inhibition of PI3K-III phosphorylation.