Gene cloning and enzymatic activity analysis of phenylalanine ammonia-lyase from Sinopodophyllum hexandrum (Royle) Ying / 生物工程学报

Phenylalanine ammonia-lyase (PAL) is the key entry enzyme of plant phenylpropanoid pathway. It plays an important role in the biosynthesis of podophyllotoxin, an anti-tumor lignan that is currently produced from its main natural source Sinopodophyllum hexandrum (Royle) Ying. In this study, we cloned the gene ShPAL encoding phenylalanine ammonia-lyase by RT-PCR from the root of S. hexandrum ecotype inhabited in the Aba' district, Sichuan, based on its public SRA transcriptome data-package. Bioinformatics analyses showed that the ShPAL-encoded protein is composed of 711 amino acids, contains the conserved domains of PAL, and has the signature motif within the active center of aromatic ammonia-lyases. Moreover, ShPAL protein was predicted to have a secondary structure mainly composed of α-helix and random coil, a typical 'seahorse' shape monomer tertiary structure, and a homologous tetramer three-dimensional structure by Swiss-Modelling. The phylogenetic lineage analysis indicated ShPAL was of the highest sequence identity and the shortest evolutionary distance with the PAL of Epimedium sagittatum from the same Berberidaceae family. Subcellular localization experiments showed that ShPAL protein was mainly distributed in the cytoplasm, despite of a minority on the endoplasmic reticulum membrane. Furthermore, ShPAL protein was recombinantly expressed in Escherichia coli and purified by histidine-tag affinity chromatography. Its enzymatic activity was determined up to 20.91 U/mg, with the optimum temperature of 41 ℃ and pH of 9.0. In contrast, the enzyme activity of its F130H mutant decreased by about 23.6%, yet with the same trends of change with temperature and pH, confirming that phenylalanine at this position does affect the substrate specificity of PAL. Both the wild type and the mutant have relatively poor thermostability, but good pH-stability. These results may help to further investigate the regulatory role of PAL in the process of podophyllotoxin biosynthesis and advance the heterologous synthesis of podophyllotoxin to protect the germplasm resource of S. hexandrum. They also demonstrate that ShPAL has a potential application in biochemical industry and biomedicine.

Effects of Tongxie-Yaofang on visceral sensitivity and expression of 5-HT and SERT in IBS-D model rats / 国际中医中药杂志

Objective:To explore the effect of Tongxie-Yaofang on visceral sensitivity in IBS-D and the possible mechanism. Methods:Divided 30 male SD rats (one-day old) into normal group (10 rats) and IBS-D model group (20 rats) randomly. IBS-D was induced by the method of neonatal maternal separation and restraint stress. After successful modeling, the IBS-D model group was randomly divided into model group and Tongxie-Yaofang group, with 10 rats in each group. Tongxie-Yaofang group was given Tongxie-Yaofang formula, 4.92 g/ml by gavage, while the normal and model groups were given the same amount of normal saline, rats were gavaged with 2 ml/100 g body weight once a day for 14 days. The electromyography of the exorectus muscle was used to meature colorectal distension and by using electronic constant pressure apparatus to evaluate visceral sensitivity. The morphology of colon by HE staining and Enzyme-linked immunosorbent assay (ELISA) were used to determine the level of colonic 5-hydroxytryptamine (5-HT), qPCR was used to detect the colonic mRNA expression of serotonin transporter (SERT) and Western blot was used to detect SERT expression in colon and hypothalamus. Results:Compared with the model group, at the expansion pressure of 60 mmHg and 80 mmHg, the electromyographic response [(179.51 ± 18.26)% vs. (226.42 ± 25.78)%; (242.13 ± 15.42)% vs. (306.02 ± 51.51)%] in Tongxie-Yaofang group was significantly decreased ( P<0.05 or P<0.01). The colonic content of 5-HT was significantly lower than that in the model group [(8.85 ± 0.53) ng/mg vs.(12.25 ± 1.95) ng/mg] ( P<0.01), the expression of SERT mRNA (0.85 ± 0.12 vs. 0.38 ± 0.02) and SERT protein (0.53 ± 0.11 vs. 0.36 ± 0.17) in the colon was significantly increased ( P<0.05 or P<0.01), the expression of SERT protein (0.88 ± 0.12 vs. 0.36 ± 0.13) in the hypothalamus was significantly increased ( P<0.05). Conclusion:Tongxie-Yaofang could relieve the visceral hypersensitivity, which may be achieved by regulating the 5-HT and SERT expression.

Effect of telmisartan on expression of metadherin in the kidney of mice with unilateral ureter obstruction / 南方医科大学学报

OBJECTIVE@#To explore the effect of telmisartan on the expression of metadherin in the kidney of mice with unilateral ureter obstruction.@*METHODS@#Eighteen male C57 mice were randomized into sham-operated group, model group and telmisartan treatment group. In the latter two groups, renal interstitial fibrosis as the result of unilateral ureter obstruction (UUO) was induced by unilateral ureteral ligation with or without telmisartan intervention. Renal pathological changes of the mice were assessed using Masson staining, and immunohistochemistry and Western blotting were used to detect the expression of extracellular matrix proteins and metadherin in the kidney of the mice. In the experiment, cultured mouse renal tubular epithelial cells (mTECs) were stimulated with transforming growth factor-β1 (TGF-β1) and transfected with a siRNA targeting metadherin, and the changes in the expressions of extracellular matrix proteins and metadherin were detected using Western blotting.@*RESULTS@#The expressions of extracellular matrix proteins and metadherin increased significantly in the kidney of mice with UUO ( < 0.05). Intervention with telmisartan significantly lowered the expressions of extracellular matrix proteins and metadherin and alleviated the pathology of renal fibrosis in mice with UUO ( < 0.05). In cultured mTECs, siRNA-mediated knockdown of metadherin obviously reversed TGF-β1-induced increase in the expressions of extracellular matrix proteins and metadherin.@*CONCLUSIONS@#Telmisartan can suppress the production of extracellular matrix proteins and the expression of metadhein to attenuate UUO-induced renal fibrosis in mice.

Identification of a novel COL2A1 variant in a pedigree affected with spondyloepiphyseal dysplasia congenita / 中华医学遗传学杂志

OBJECTIVE@#To explore the molecular basis for a pedigree affected with spondyloepiphyseal dysplasia congenita (SEDC).@*METHODS@#The proband was subjected to whole exome sequencing. Suspected variant was verified by Sanger sequencing.@*RESULTS@#All patients from the pedigree were found to carry a novel missense variant c.1394G>C (p.Gly465Ala) of the COL2A1 gene. The variant was not reported previously. Provean, Polyphen-2 and Mutation Taster software predicted that the variant is highly likely to be pathogenic.@*CONCLUSION@#The c.1394G>C (p.Gly465Ala) variant of the COL2A1 gene probably underlies the SEDC in this pedigree.

Evaluation of left atrial function in patients with severe preeclampsia by full-volume imaging technology / 中华超声影像学杂志

Objective To evaluate the left atrial(LA) function in patients with severe preeclampsia ( SPE) by full-volume imaging technology. Methods Forty-two patients of SPE were randomly chosen as SPE group,34 healthy pregnant women were selected as the control group. LA related function parameters including LA maximum volume( LAVmax),LA minimum volume( LAVmin),LA pre-atrium contraction volume(LAVpre),LA reservoir volume(LARV),LA passive emptying volume(LAPEV),LA contraction volume(LACV),LA expansion index(LAEI),LA passive emptying fraction(LAPEF),LA passive emptying fraction( LAAEF ) were separately required by biplane area-length method and full-volume imaging technology in antepartum and postpartum. Results Compared with control group before delivery,the parameters of SPE group before delivery such as LAVmax,LAVmin,LAVpre were higher,LAEI,LAPEF, LAAEF indexed to body surface area(BSA) were lower(all P ＜0.01).Compared with control group before delivery,the parameters of control group after delivery such as LAVmax,LAVmin,LAVpre were lower( all P ＜0.05),LAEI, LAAEF calculated as ratio to BSA were somewhat reduced,LAPEF calculated as ratio to BSA was relatively higher(all P ＞0.05).Compared with SPE group before delivery,the parameters of SPE group after delivery such as LAVmax,LAVmin,LAVpre were lower,LAEI,LAPEF,LAAEF indexed to BSA were higher( all P ＜0.01). Compared with control group after delivery,the parameters of SPE group after delivery such as LAVmax,LAVmin,LAVpre were still incresed ( all P ＜ 0.05 ), LAEI,LAPEF, LAAEF indexed to BSA were relatively lower( all P ＞0.05). Conclusions Full-volume imaging technology can quantitatively evaluate the LA function. LA reservoir function,conduit function and booster pump function are reduced in SPE before delivery,LA rereservoir function,conduit function and booster pump function are basically recovery in SPE after delivery.

Feasibility of Gas as A Contrast Agent for Stomach MRI Examination / 中国医学影像学杂志

PurposeGas is rarely studied or reported to be used as a contrast agent of stomach MRI examination because it might lead to susceptibility artifacts, the purpose of this study is to evaluate the application value of gas as a contrast agent of stomach MRI examination.Materials and Methods Sixty patients who were supposed to have upper abdominal MRI examination were enrolled, all patients were given intramuscular injection of 654-2, gas from oral ingestion of aerogenic powder was used as contrast agent, MRI examinations were then executed and images quality of MRI were analyzed and classified. Results All 60 patients felt it acceptable for the taste of aerogenic powder, only 5 cases felt uncomfortable during swallowing; no adverse reactions were observed in all the 60 cases. Excellent gas contrast medium filling of the stomach cavity were confirmed with CT scanning in 57 cases (95%). Gas acted as a negative contrast agent in the stomach cavity with good continuity and uniform signal; and the proximal part of the stomach and duodenum was well displayed. There were 21 cases (35.0%) classified as grade 0 about their image quality; 25 cases (41.7%) as grade 1; 6 cases (10.0%) as grade 2; 8 cases (13.3%) as grade 3, with total 52 cases (86.7%) with image quality which could meet the requirement of clinical diagnosis. Cases of grade 0-2 showed coronary T2WI images with vivid stomach contour and clear stomach wall with no artifacts; axial FS-T2WI images with few artifacts, relative clear stomach contour and good display of stomach wall; less artifact could be found in axial TIWI image with good display of stomach wall; axial T2WI images showed more artifacts and gastric wall was displayed not clear enough. Cases classified as grade 3 showed coronal T2WI, FS-T2WI, T1WI and T2WI axial images with significant artifacts, vague outline, distortions or ghosting of the contour of stomach and tumor.Conclusion As contrast agent for gastric MRI examination, gas is safe, homogeneously distributed with stable signal characteristic and is easy for patients to tolerate.

Intradiscal injection of ozone combined with epidural injection of collagenase for the treatment of prominent lumbar disc protrusion: a multicentral application observation / 介入放射学杂志

Objective To investigate the curative effect and safety of percutaneous intradiscal injection of ozone combined with epidural injection of collagenase for the treatment of prominent lumbar disc protrusion.Methods A total of 541 patients with lumbar disc protrusion were included in this study.All the patients fulfilled the following criteria:the fibrous ring was broken,the protruded extent of nucleus pulposus was less than 10 mm and the dura sac and/or nerve root was compressed.The intradiscal injection of ozone combined with epidural injection of collagenase was performed in all patients.The patients were followed up through further consultation,letters,telephone or other ways.Five hundred and forty-one cases were followed up for 2-3 months(short-term),312 cases for 4-12 months(medium-term)and 115 cases for 13-18 months(ling-term).The short-term,medium-term and long-term curative effects were evaluated with unified curative criteria,and the results were compared with that obtained with the treatment of epidural injection of collagenase only. Results The short-term, medium-term and long-term effective rate of selected cases was 95.9%(519/541),90.4%(489/541)and 87.2%(472/541),respectively.When combined application of two methods was performed,different degree of shrinkage of the protruded nucleus pulposus was obviously observed,with a mean shrinkage degree of 30.5%.The short-term,medium-term and long-term effective rate of simple collagenase chemonucleolysis was 89.5%,82.4%and 80.4%,respectively.Conclusion Intradiscal injection of ozone combined with epidural injection of collagenase is an effective treatment for lumbar disc protrusion when the fibrous ring is broken,the protruded extent of nucleus pulposus is less than 10 mm and the dura sac and/or nerve root is compressed. Its curative effect is superior to simple collagenase chemonucleolysis. Ozone is of clinical significance for producing the shrinkage of the nucleus pulposus.

Treatment of Limb Pain after Wind-stroke by Direct Moxibustion with Small Moxa Cones / 针灸推拿医学（英文版）

Thirty-one cases of limb pain after wind-stroke were treated by direct moxibustion with small moxa cones. Results showed that 12 cases got remarkable effects, 15 cases effect and 4 cases ineffectiveness, with a total effective rate of 87.1%.

Intradiscal injection of ozone combined with epidural injection of collagenase for the treatment of prominent lumbar disc protrusion:a multicentral application observation / 介入放射学杂志

Objective To investigate the curative effect and safety of percutaneous intradiscal injection of ozone combined with epidural injection of collagenase for the treatment of prominent lumbar disc protrusion.Methods A total of 541 patients with lumbar disc protrusion were included in this study.All the patients fulfilled the following criteria:the fibrous ring was broken,the protruded extent of nucleus pulposus was less than 10 mm and the dura sac and/or nerve root was compressed.The intradiscal injection of ozone combined with epidural injection of collagenase was performed in all patients.The patients were followed up through further consultation,letters,telephone or other ways.Five hundred and forty-one cases were followed up for 2-3 months(short-term),312 cases for 4-12 months(medium-term)and 115 cases for 13-18 months(long-term).The short-term,medium-term and long-term curative effects were evaluated with unified curative criteria,and the results were compared with that obtained with the treatment of epidural injection of collagenase only.Results The short-term,medium-term and long-term effective rate of selected cases was 95.9%(519/541),90.4%(489/541)and 87.2%(472/541),respectively.When combined application of two methods was performed,different degree of shrinkage of the protruded nucleus pulposus was obviously observed,with a mean shrinkage degree of 30.5%.The short-term,medium-term and long-term effective rate of simple collagenase chemonucleolysis was 89.5%,82.4%and 80.4%,respectively.Conclusion Intradiscal injection of ozone combined with epidural injection of collagenase is an effective treatment for lumbar disc protrusion when the fibrous ring is broken,the protruded extent of nucleus pulposus is less than 10 mm and the dura sac and/or nerve root is compressed.Its curative effect is superior to simple collagenase chemonucleolysis.Ozone is of clinical significance for producing the shrinkage of the nucleus pulposus.