ABSTRACT
This paper aimed to study the role of asparagine endopeptidase(AEP) gene in the biosynthesis mechanism of cyclic peptide compounds in Pseudostellaria heterophylla. The transcriptome database of P. heterophylla was systematically mined and screened, and an AEP gene, tentatively named PhAEP, was successfully cloned. The heterologous function verification by Nicotiana benthamiana showed that the expression of the gene played a role in the biosynthesis of heterophyllin A in P. heterophylla. Bioinformatics analysis showed that the cDNA of PhAEP was 1 488 bp in length, encoding 495 amino acids with a molecular weight of 54.72 kDa. The phylogenetic tree showed that the amino acid sequence encoded by PhAEP was highly similar to that of Butelase-1 in Clitoria ternatea, reaching 80%. The sequence homology and cyclase active site analysis revealed that the PhAEP enzyme may specifically hydrolyse the C-terminal Asn/Asp(Asx) site of the core peptide in the HA linear precursor peptide of P. heterophylla, thereby participating in the ring formation of the linear precursor peptide. The results of real-time quantitative polymerase chain reaction(RT-qPCR) showed that the expression level of PhAEP was the highest in fruits, followed by in roots, and the lowest in leaves. The heterophyllin A of P. heterophylla was detected in N. benthamiana that co-expressed PrePhHA and PhAEP genes instantaneously. In this study, the PhAEP gene, a key enzyme in the biosynthesis of heterophyllin A in P. heterophylla, has been successfully cloned, which lays a foundation for further analysis of the molecular mechanism of PhAEP enzyme in the biosynthesis of heterophyllin A in P. heterophylla and has important significance for the study of synthetic biology of cyclic peptide compounds in P. heterophylla.
Subject(s)
Genes, vif , Phylogeny , Plant Leaves/genetics , Peptides, Cyclic , Cloning, Molecular , Caryophyllaceae/geneticsABSTRACT
Objective:To study on the correlation between integrated pharmacokinetics and pharmacodynamic effects of five active components(oxidized paeoniflorin,paeoniflorin,quercetin,gallic acid, paeonol) in Moutan Cortex. Method:Rats were divided into blank group,model group(syndrome of blood-heat and blood stasis) and drug-administered group.The concentration of five active components in serum were detected with UPLC-MS at different time points after being administrated ethanol extract of Moutan Cortex.The integrated concentrations were calculated according to area under the curve(AUC) self-defined weighting coefficiency.At the same time,the enzyme-linked immunosorbent assay(ELISA) was used to determine the contents of thromboxane B2(TXB2) and 6-keto-prostaglandin F1α(6-keto-PGF1α) in serum at different time points,and then correlation between pharmacodynamics and integrated pharmacokinetics of these five active ingredients was analyzed. Result:At different time points(0.083,0.25,0.5,0.75,1,2,3,4,6,8,10,12 h),the integrated plasma concentrations of these five active ingredients in Moutan Cortex(158.65,174.60,220.13,227.23,244.31,251.51,404.28,654.39,472.62,355.04, 231.56,199.40 mg·L-1) had a good correlation with concentration of TXB2(264.44,261.03,284.93,273.30,264.04, 278.90,274.83,303.58,260.03,264.78,264.40,256.62 μg·L-1) and value of TXB2/6-keto-PGFlα(4.50,4.47,3.66,3.37, 3.29,3.66,3.71,4.30,3.63,3.65,3.75,3.66). Conclusion:There is a good correlation between the dynamic changes in vivo of active components from Moutan Cortex and pharmacodynamic effects of activating blood circulation of this herb.
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Objective To determine the feasibility of neck vessel wall imaging technology with three?dimensional variable?flip?angle turbo spin?echo (3D T1w?SPACE) for the detection of carotid atherosclerotic disease before revascularization. Methods Thirty?one patients who underwent carotid endarterectomy (CEA) and fifty?three patients who underwent carotid stenting (CAS) were enrolled prospectively. Neck vessel wall imaging examination were performed in all patients whilecarotid artery DSA were performed in all CAS patients. Quantitative measurements including stenosis, lesion length, and the presence or absence of plaque ulceration obtained with 3D T1w?SPACE and DSA were independently determined. And images of the 3D T1w?SPACE were compared with corresponding histology to identify major plaque components including intraplaque hemorrhage (IPH), lipid rich necrotic core (LRNC), and calcification (CA). The consistency rate, sensitivity, specificity, positive predictive value and negative predictive value were used to assess diagnostic value. Bland?Altman plots, intraclass correlation coefficient (ICC), and Cohen Kappa were determined. Results DSA was served as the reference standard. There was an excellent correlation between 3D T1w?SPACE and DSA images in measuring stenosis (r=0.984, P<0.01) and luminal stenosis [ICC=0.98 (95% confidence interval: 0.96-0.99)]. Bland?Altman plots showed that the two examinations were in good consistency in evaluating the extent of stenosis. Sensitivity (89.5%) and specificity (95.1%) was high in 3D T1w?SPACE images compared to DSA for the detection of ulcers. The consistency rate between 3D T1w?SPACE images and histological results for IPH, LRNC and CA detection were 85.7%, 82.1% and 92.9%, respectively. Sensitivity and specificity were 90.0% and 75.0% for IPH;83.3% and 80.0% for LRNC; 91.3% and 100.0% for CA respectively. However, lesion length measurements by using 3D T1w?SPACE were longer than those measured by using DSA (P<0.01).Conclusion Neck vessel wall imaging technology with 3D T1w?SPACE is a noninvasive and accurate technique for the diagnosis of carotid artery atherosclerotic disease before revascularization.
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To observe the effect of intaspinal implantation of Schwann cells (SC) genetically modified with microgene pSVPoMcat on spinal cord injury (SCI) repair.120 SD rats were used to establish the hemisected spinal cord injury model at T 8 level,and they were divided randomly into three groups: genetically modified SC implantation group (group A),normal SC implantation group (group B) and control group without cell implantation (group C).One week after the operation ,combined behavioral score(CBS) and the cortical somatasensory evoked potential (GFAP) were measured and the expression of glial fibrillary acidic protein(GFAP) was examined by in situ hybridization and immunocytochemistry.Three months after the operation, all the rats were scanned with MRI and then were sacrificed.Neurofilament (NF) was examined with imunohistocytochemistry staining by using NF monoclonal antibody. Following were the results:(1) In group A,the number of cells expressed GFAP in injured sites was less than that in groups B and C.(2) MRI scanning showed that the SCI region almost recovered in group A but did not recover in group B.There was a malacie focus in SCI region in group C.This was corroborated by the NF staining.(3) The amplitudes of potential in the latent period in group A and B showed a tendency to recover,and it was consistent with CBS.The results suggested that the implantation of genetically modified SC with microgene pSVPoMcat could inhibit GFAP expression and promote the functional recovery of spinal cord injury in rats.