Objective Deleting the cysteine-rich region (22-37 amino acids)of HIV-1 HXB2 Tat protein(whole length is 101 amino acids) to improve its stability and expression level in E.coli and to analyze the immunogenicity of Tat protein without the cystein-rich region [Tat(△C)protein]. Methods Tat DNA deleted the cysteine-rich region (64-111 nucleotides), named as Tat(△C)DNA, was obtained in vitro by PCR and cloned into pET-32a vector. pET-32a-Tat(△C)plasmid and the pET-32a-Tat plasmid were established and transformed into E.coli BL21(DE3) strains respectively to express and purify the protein. Three rabbits were vaccinated with pET-32a-Tat(△C)protein, then testify the reactivity of sera from rabbits by ELISA and Western blot. Results The dense of the purified pET-32a-Tat(△C)protein was 7.12 mg/ml,which was greatly more than pET-32a-Tat protein(1.50 mg/ml). Dimer of pET-32a-Tat protein can be observed just after the protein purification and stored at 25℃ and 4℃ for 7 days, but dimer of pET-32a-Tat(△C)protein was not formed at the same condition. Experimental rabbits were immunized with pET-32a-Tat(△C)protein and produced high titre of anti-pET-32a-Tat(△C)serum(1∶320 000), the antibody can react specifically with Tat(△C)protein, Tat protein (1-101 AA)and synthetic Tat(1-86 AA) protein. Deletion mutation of the cysteine-rich region of Tat protein was first performed in the study. Conclusion The expression level in E.coli and the stability of Tat protein deleted the cysteine-rich region can be increased greatly, and the protein remains good immunogenicity. The results may provide a novel antigen for further development of HIV-1 Tat vaccine.

OBJECTIVE: To study the mechanisms of Tongxia Huayu Decoction (a Chinese herbal decoction for purgation and removing blood stasis) in prognostic improvement for severe acute pancreatitis by early intervention on pancreatic microcirculatory disturbance. METHODS: Fifty-three patients with severe acute pancreatitis were divided randomly into treatment group (n=28) and control group (n=25). Tongxia Huayu Decoction was given to the patients in treatment group in addition to the normal treatment in control group for one week. The clinical symptoms and signs, hemodiastase, urinary amylase, C-reactive protein (CRP) and endothelin (ET) of the patients in the two groups before and after treatment were observed and detected. RESULTS: The total response rate of the treatment group was 98.4%, while that of the control group was 80%, with significant difference between them (P<0.05). There was no significant difference of the contents of hemodiastase, urinary amylase, CRP and ET between the two groups before treatment, while they were significantly decreased after treatment (P<0.01) with more obvious change in treatment group (P<0.01). CONCLUSION: Tongxia Huayu Decoction brings satisfied therapeutic effect on severe acute pancreatitis. The mechanisms may associate with its reducing function on ET releasing so as to inhibit the pancreatic microcirculatory disturbance and acinar cell injury induced by ET.

OBJECTIVETo study the alkaloids of the Chinese medicinal herbs Daphniphyllum macropodum for in search for new bioactive substances.

METHODThe CHCl3 extract of D. macropodum was subjected to repeated column chromatography on silica gel to afford four pure compounds (1-4). Their structures were determined on the basis of detailed spectroscopic analysis and comparison with the data reported in the literature.

RESULTThe four known compounds were isolated and elucidated as Daphniphyllum alkaloids with different carbon skeletons, namely longistylumphylline A (1), deoxycalyciphylline B (2), daphnicyclidin B (3) and H (4), respectively.

CONCLUSIONAll compounds were isolated from the titled plant for the first time. The discovery of daphnicyclidin B (3) and H (4) further confirmed the biogenetic relationship between the two compounds and the previously reported macropodumines A-C, found in the same plant.

A 1 270 bp full-length cDNA fragment was obtained from the Schistosoma japonicum (Chinese strain) adult cDNA library after the '3' and 5' ends of the incomplete expression sequence tag (EST) of hypoxanthine-guanine phosphoribosyltransferase of Schistosoma japonicum (SjHGPRT) were amplified by the anchored PCR with 2 pairs of primer that were designed according to the published incomplete SjHGPRT EST and the sequence of multiclone sites of library λgt1 1 vector. Sequence analysis indicated that this fragment, with an identity of 82% to hypoxanthine-guanine phosphoribosyltransferase ofSchistosoma mansoni (SmHGPRT), contained a complete open reading frame(ORF). The deduced amino acid sequence showed 83% identity to that of SmHGPRT. This fragment was cloned into the prokaryotic expression vector pQE30, and subsequently sequenced and expressed in Escherichia coli. SDS-PAGE revealed that M of the recombinant protein was about 28 ku. Western-blot analysis showed that the recombinant protein was recognized by the polyclonal antisera from rabbits immunized with Schistosoma japonicum adult worm antigen. Mice vaccinated with recombinant protein revealed significant worm burden, liver eggs per gram (LEPG), fecal eggs per gram (FEPG) and intrauterine eggs of the female worms reduction percentage, compared with the controls. Taken together, the SjHGPRT full-length cDNA can be cloned and expressed in E. coli as a recombinant protein that elicited immunity against the challenge infection with Schistosoma japonicum, indicating its potential as a partia1 protection vaccine candidate.

A 1 270 bp full-length cDNA fragment was obtained from the Schistosoma japonicum (Chinese strain) adult cDNA library after the 3′ and 5′ ends of the incomplete expression sequence tag (EST) of hypoxanthine-guanine phosphoribosyltransferase of Schistosoma japonicum (SjHGPRT) were amplified by the anchored PCR with 2 pairs of primer that were designed according to the published incomplete SjHGPRT EST and the sequence of multiclone sites of library ?gt11 vector. Sequence analysis indicated that this fragment, with an identity of 82% to hypoxanthine-guanine phosphoribosyltransferase of Schistosoma mansoni (SmHGPRT), contained a complete open reading frame(ORF). The deduced amino acid sequence showed 83% identity to that of SmHGPRT. This fragment was cloned into the prokaryotic expression vector pQE30, and subsequently sequenced and expressed in Escherichia coli. SDS-PAGE revealed that M of the recombinant protein was about 28 ku. Western-blot analysis showed that the recombinant protein was recognized by the polyclonal antisera from rabbits immunized with Schistosoma japonicum adult worm antigen. Mice vaccinated with recombinant protein revealed significant worm burden, liver eggs per gram (LEPG), fecal eggs per gram (FEPG) and intrauterine eggs of the female worms reduction percentage, compared with the controls. Taken together, the SjHGPRT full-length cDNA can be cloned and expressed in E.coli as a recombinant protein that elicited immunity against the challenge infection with Schistosoma japonicum, indicating its potential as a partial protection vaccine candidate.

OBJECTIVETo evaluate the difference in the clinical therapeutic effects on anal fissure at Ⅰ and Ⅱ stages between the acupoint catgut embedding therapy and western medication.

METHODSSixty patients of anal fissure at Ⅰ and Ⅱ stages were randomized into an embedding therapy group and a western medication group, 30 cases in each one. In the embedding therapy group, the acupoint catgut embedding therapy was applied at bilateral Tianshu (ST 25), Changqiang (GV 1), bilateral Chengshan (BL 57) and Tigangxue (Extra), once a week. In the western medication group, the external inunctum on the wound was given with 0.2% nitroglycerin ointment, once every morning and evening a day. The treatment lasted for 4 weeks continuously in the two groups. The follow-up visit was done for 3 months after treatment. The visual analogue scale (VAS) and anal pain duration were observed and recorded before treatment and on the 3rd day and the 7th day of treatment separately. The clinical therapeutic effects were compared between the two groups.

RESULTSAfter treatment, on the 3rd day and the 7th day of treatment, VAS score and anal pain duration were all reduced significantly as compared with those before treatment in the patients of the two groups (all<0.01). The differences in the embedding therapy gruop were better than those in the western medication group before and after treatment (<0.01,<0.05). In the 2nd and 4th weeks after treatment, the clinical therapeutic effects in the embedding therapy group were better than those in the western medication group (both<0.05). In 3-month follow-up, the recurrent case in the embedding therapy group was one, and the recurrent case in the western medication group was six.

CONCLUSIONSThe acupoint catgut embedding therapy is safe and effective in the treatment of anal fissure at Ⅰ and Ⅱ stages and its recurrent case is lower as compared with the treatment of western medication.

Objective:To establish a method for quality evaluation of Myristica fragrans Houtt. Methods:The common peak was determined with Dehydroisoeugenol as the reference peak, and the HPLC fingerprint of Myristica fragrans was established; then the common peaks were analyzed by High Resolution Liquid Chromatography-mass Spectrometry (HPLC-MS). The chemical components of the common peaks were identified through the calculation and data retrieval of the primary and secondary mass spectra of the characteristic peaks. Results:The HPLC fingerprint of Myristica fragrans was established, and the similarity degree of the 10 batches of samples was above 0.9 and 11 common peaks were established. According to the results of HPLC-MS, the components of 11 common peaks were identified as follow: Methyl eugenol (peak 1), Licarin A (peak 2), Myristol (peak 3), OdoratisolA (peak 4), 2-(3,4-Dimethoxyphenyl) butynoic acid (peak 5), Malabaricon D (peak 6), 5'-Methoxydehydroisoeugenol (peak 7), Dehydroisoeugenol (peak 8), Malabaricone C (peak 9), 4-Methoxy-6-{(2S,3S)-7-methoxy-3-methyl-5-[(1E)-1-propen-1-yl]-2,3-dihydro-1- benzofuran-2-yl}-1,3-benzodioxole (peak 10) and Licarin B (peak 11). Conclusions:The quality of Myristica fragrans could be evaluate with HPLC fingerprint method. HPLC-MS was used to analyze the chemical composition of complex components, which will provide reference for the identification and analysis of chemical components of the extracts and preparations of Traditional Chinese Medicine.

OBJECTIVE：To establish a m ethod for simultaneous determination of neoastilbin ，astilbin，neoisoastilbin，isoastilbin， quercitrin and engeletin in Engelhardia roxburghiana，and conduct multivariate statistical analysis. METHODS ：HPLC-QAMS method was adopted. The determination was performed on Phenomenex SuperLu C 18 column with mobile phase consisted of acetonitrile-0.1％ formic acid （19 ∶ 81，V/V）at the flow rate of 1.0 mL/min. The detection wavelengths were set at 254 nm （neoastilbin，astilbin，neoisoastilbin，isoastilbin，engeletin）and 291 nm（quercitrin）. The column temperature was 30 ℃，and sample size was 10 μL. Using astilbin as internal substance，and the relative correction factors of other 5 factors were calculated. The contents of each component were calculated according to relative correction factor ，and were compared with the results of external standard method. SPSS 22.0 software was used for cluster analysis and principal component analysis. RESULTS ：The linear range of neoastilbin ，astilbin，neoisoastilbin，isoastilbin，quercitrin and engeletin were 0.007-0.311，0.871-18.184，0.002-0.119， 0.052-1.251，0.105-2.202，0.020-2.319 μg（r＞0.999），respectively. RSDs of precision ，reproducibility and stability （24 h）tests were all lower than 3％. The average recoveries were 97.32％，94.89％，97.15％，96.90％，97.52％ and 97.53％（RSDs were 1.09％ -2.60％ ，n＝6），respectively. The relative correction factors of neoastilbin ，neoisoastilbin，isoastilbin，quercitrin and engeletin were 1.252 6，1.198 3，0.958 6，0.807 1 and 1.138 1， respectively. The contents of neoastilbin ， neoisoastilbin， qq.com isoastilbin，quercitrin and engeletin measured by QAMS were 0.394 2-2.067 2，0.139 1-0.804 7，2.864 8-8.554 8，4.581 2- 11.371 1，1.028 9-13.401 5 mg/g；the contents of neoastilbin ， astilbin，neoisoastilbin，isoastilbin，quercitrin and engeletin were 0.367 2-1.925 3，46.361 1-126.342 1，0.138 1-0.798 8，2.966 2-8.857 8， 4.642 5-11.523 3，0.970 6-12.641 9 mg/g，respectively. Relative errors of two methods was lower than or equal to 3.55％. The results of cluster analysis showed that 9 batches of samples could be clustered into two categories ；S8 sample was one category and others were one category. The results of principal component analysis showed that accumulative contribution rate of former 2 principle components was 84.745％，and the results of sample classification were consistent with those of cluster analysis. CONCLUSIONS ： The established HPLC-QAMS method is accurate ，feasible and repeatable ，and can be used for simultaneous determination of 6 flavonoids in E. roxburghiana ，and it can provide reference for quality control.

Marsdenia tenacissima injection, a standard Marsdenia tenacissima extract (MTE), has been approved as an adjuvant therapeutic agent for various cancers. Our previous study showed that MTE inhibited the proliferation and metastasis of prostate cancer (PCa) cells. However, the underlying mechanisms and active ingredients of MTE against PCa were not completely understood. This study revealed that MTE induced significant decreases in cell viability and clonal growth in PCa cells. In addition, MTE induced the apoptosis of DU145 cells by reducing the mitochondrial membrane potential and increasing the expression of Cleaved Caspase 3/7, Cyt c, and Bax. In vivo, DU145 xenografted NOD-SCID mice treated with MTE showed significantly decreased tumor size. TUNEL staining and Western blot confirmed the pro-apoptotic effects of MTE. Network pharmacology analysis collected 196 ingredients of MTE linked to 655 potential targets, and 709 PCa-associated targets were retrieved, from which 149 overlapped targets were screened out. Pathway enrichment analysis showed that the HIF-1, PI3K-AKT, and ErbB signaling pathways were closely related to tumor apoptosis. Western blot results confirmed that MTE increased the expression of p-AKT^Ser473 and p-GSK3β^Ser9, and decreased the expression of p-STAT3^Tyr705in vitro and in vivo. A total of 13 compounds in MTE were identified by HPLC-CAD-QTOF-MS/MS and UPLC-QTOF-MS/MS. Molecular docking analysis indicated that six compounds may interact with AKT, GSK3β, and STAT3. In conclusion, MTE induces the endogenous mitochondrial apoptosis of PCa by regulating the AKT/GSK3β/STAT3 signaling axis, resulting in inhibition of PCa growth in vitro and in vivo.
Mice ; Animals ; Male ; Humans ; Mice, Inbred NOD ; Mice, SCID ; Marsdenia ; Proto-Oncogene Proteins c-akt ; Glycogen Synthase Kinase 3 beta ; Molecular Docking Simulation ; Phosphatidylinositol 3-Kinases ; Tandem Mass Spectrometry ; Prostatic Neoplasms ; Apoptosis ; STAT3 Transcription Factor