2.Cloning and eukaryotic expression of human CD38
Xinyu WEN ; Cuiling SHU ; Yan LI ; Zhongtian QI ; Beife SHEN
Academic Journal of Second Military Medical University 2001;0(09):-
Objective:To clone and express the full length cDNA of human CD38. Methods:The full length cDNA of the human CD38 antigen was amplified from total RNA of Daudi cell by RT-PCR, and it was inserted into pGEM-T. The validity on the sequences was confirmed by automatic DNA sequencing. Inserting the valid CD38 gene into pcDNA3.1(+) plasmid to obtain recombinant mammalian expression vector pcDNA3.1(+)/CD38Z; Using lipofectin gene transfer technique system, recombinant expression vector containing CD38 gene was transfected into COS7 cells. The expression of CD38 molecules on the surface of COS7 cells was detected by FACS and immunohistochemical technique. Results:DNA sequencing showed that the cloned full length cDNA sequence was identical with reported. The result of FACS and immunohistochemical technique indicated that CD38 molecules were expressed on the surface of COS7 cells. Conclusion:The full length cDNA of human CD38 is obtained, recombinant mammalian expression vector pcDNA3.1(+)/CD38Z is successfully constructed, and the CD38 molecules is expressed on the surface of COS7 cells,this may facilitate studies on the biochemistry and function of CD38 antigen.
3.Optimization of Purification and Isolation of Punicalagin from Pomegranate Peel
Shu LIU ; Wanli SHEN ; Guan LIAN ; Wen CHEN ; Rong QI
China Pharmacist 2017;20(1):14-19
Objective: To establish the methods for extraction and isolation of punicalagin from pomegranate peel, and further study the purification and quantification of punicalagin. Methods: Using an ultrasonic-assisted extraction method, punicalagin in pomegranate peel was extracted at room temperature by 50% ethanol with 20-fold volume of raw material. The content of punicalagin in the crude extract was determined by HPLC. To optimize the purification process of punicalagin, static adsorption and desorption experi-ments were employed to study five kinds of macroporous adsorbent resins (D101, A8-8, NKA-9, HPD-100 and HPD-500) for the one with the highest purification efficacy of punicalagin. In addition, the technical parameters of the macroporous adsorbent resin were opti-mized to obtain punicalagin with higher purity. Punicalagin was further separated and purified by using a reverse phase MCI GEL CHP20P column. Results:HPD500 resin showed the best ability to absorb and separate punicalagin in among five kinds of macro-porous adsorbent resins. The best technical parameters were as follows:the mass concentration of sample solution was 15 mg·ml-1 , the loading amount was 2BV, the pH was 2 and the eluting solvent was 8BV of 30% ethanol. With the best process as described a-bove, the content of punicalagin extracted from pomegranate peel increased from 10. 3% to 30. 7%. The obtained punicalagin could be further purified to 61. 3% from 30% in ethanol eluate by the reverse phase MCI GEL CHP20P column. Conclusion:HPD500 resin is the most effective in the purification of punicalagin from pomegranate peel, and the content of punicalagin can be dramatically increased after the purification by a reverse phase MCI GEL CHP20P column. The optimized process shows good reproducibility and stability.
4.Analysis of Newborn′s Weight Investigation in Some Areas of Henan Province
xiao-shu, LI ; hu, ZHAO ; wen-juan, YIN ; ai-hong, WU ; su-fang, SHEN
Journal of Applied Clinical Pediatrics 2004;0(09):-
Objective To investigate the full-term newborn′s weight in Zhengzhou city and nearby areas around Zhengzhou in Henan province.Methods Each group newborn′s weight was divided with sex and city.We studied the regularity of full-term newborn′s weight,and examined the cause of the newborn′s weight rising.Results The average newborn′s weight in Zhengzhou was (3449.06?453.97) g,which in nearby areas around Zhengzhou was (3352.07?429.91) g.The average newborn′s weight in Zhengzhou was 86.97 g higher than other cities (P
5.A clinical study of Suogudan granule in the treatment of rheumatoid arthritis.
Wen-ya YU ; Shu-wen SHEN ; Zhi-hong YANG
Chinese journal of integrative medicine 2005;11(4):255-259
OBJECTIVETo study the clinical efficacy of Suogudan Granule (SGDG) in the treatment of rheumatoid arthritis (RA).
METHODSNinety patients with RA were randomly divided into the treated group and the control group. The treated group was administered orally with SGDG 6 g each time, thrice a day, while the control group with the combined therapy of Fenbid Capsules 0.3 g each time, twice a day and Tripterygium tablet 20 mg each time, thrice a day. The treatment course for both groups was 6 weeks. The changes of clinical symptoms and signs, and laboratory indices such as erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), antistreptolysin O (ASO), routine examination of blood and urine, liver and kidney function, etc. before and after treatment were observed.
RESULTS(1) The total effective rate in the treated group (88.0%) was obviously higher than that in the control group (67.5%) with significant difference (P < 0.05). (2) The improvement in arthralgia, joint swelling, time of morning stiffness, 15-meter walking, analgesia initiation and persistence in the treated group was better than that in the control group (P < 0.05, P < 0.01), but there was no obvious difference in improvement of joint tenderness, range of joint motion, grip strength, and initiating detumescence time (P > 0.05). (3) The improvement in ESR and RF in the treated group was better than that in the control group with significant difference (P < 0.05). The negative-conversion rate of ASO in the treated group was also higher than that in the control group (P < 0.01). (4) No evident abnormality in blood, urine, liver or kidney function was found in either group.
CONCLUSIONSGDG is effective and safe for the treatment of RA.
Administration, Oral ; Adult ; Aged ; Antistreptolysin ; analysis ; Arthritis, Rheumatoid ; drug therapy ; Blood Sedimentation ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Humans ; Male ; Middle Aged ; Phytotherapy ; Plant Preparations ; administration & dosage ; Rheumatoid Factor ; analysis ; Tripterygium
6.Quick diagnosis of glanzmann's thrombasthenia with flow cytometry.
Hai-dong FU ; Hong-qiang SHEN ; Bo-qin QIAN ; Yong-min TANG ; Hua SONG ; Shu-wen SHI ; Shi-long YANG
Chinese Journal of Pediatrics 2003;41(5):375-376
Child
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Child, Preschool
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Female
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Flow Cytometry
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methods
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Humans
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Infant
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Male
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Thrombasthenia
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classification
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diagnosis
7.Expression and Biological Characterization of Human Osteoprotegerin Fused with Mycobacteria Heat Shock Protein 65
Yue ZHANG ; Shu LIU ; Jing MA ; Shen-Tao LI ; Wei WANG ; Quan-Geng ZHANG ; Wen-Ming ZHAO ;
China Biotechnology 2006;0(04):-
A fused functional gene of human OPG and Mhsp65 was amplified by PCR,and cloned into the prokaryotic expression vector pET-28a.The BL21(DE3) strain of E.coli was transformed using the recombinant plasmid pET-28a-OPG-HSP65 and the expected protein was expressed by induction with IPTG.Result of SDS-PAGE indicated that the expected recombinant protein of 23 kDa was expressed with high yield as inclusion body.The fusion protein could be specifically recognized by both the anti-His antibody and anti-human OPG monoclonal antibody in Western blot analysis.The purified and refolded fusion protein could inhibit osteoclast proliferation and bone absorption in vitro.The results of mouse ear swelling assay and expressions of TNF-?,IFN-? and IL-17 mRNAs detected by real-time quantitative PCR demonstrated that the fusion protein had an anti-inflammation activity.The results suggest that the fusion protein of human OPG and Mhsp65 may act as a potential therapeutics for rheumatoid arthritis.
8.Effect of Paraformaldehyde Fixation on the FRET Efficiency Calculation between Fluorescent Proteins
Hong-Wei SHAO ; Wen-Feng ZHANG ; Qing-Lian HU ; Han SHEN ; Feng-Lin WU ; Shu-Lin HUANG ;
China Biotechnology 2006;0(07):-
Objective:To investigate the effect of paraformaldehyde fixation on measuring the protein-protein interaction by fluorescence resonance energy transfer(FRET)to resolve the problem of FRET efficiency calculation in excess-movement cells.Methods:The C terminals of TCR ? chain(TRA)and TCR ? chain(TRB)genes,which were ideal for protein-protein interaction research,were fused with ECFP and EYFP gene respectively by fusion PCR and transferred into target cell.A grou Pcells were fixed in paraformaldehyde(0.5%)for 0.5~1h and another left alive,then these cells were subject to ECFP/EYFP FRET calculation with confocal laser scanning microscope.The ECFP/EYFP FRET efficiencies in live and fixed cell were analyzed and compared.Results:There is no significant statistical difference between the ECFP/EYFP FRET efficiencies of live cell and cell fixed with lower paraformaldehyde concentration and shorter incubation time.Conclusion:fixation with low-concentration paraformaldehyde and short-time incubation has no distinct influence on measuring protein-protein interaction,and facilitated the FRET calculation in excess-movement cells.
9.Quantification of phenylalanine in the brain of patients with hyperphenylalaninemia by (1)H magnetic resonance spectroscopy.
Kun-di WANG ; Zhong-shu ZHOU ; Ming SHEN ; Wen HONG ; Wei-min YU
Chinese Journal of Pediatrics 2009;47(2):119-123
OBJECTIVEHyperphenylalaninemia (HPA) is an inborn error of metabolism in which the hydroxylation of phenylalanine (Phe) to tyrosine is disturbed. Accumulation of Phe leads to severe mental and psychomotor retardation. (1)H magnetic resonance spectroscopy ((1)HMRS) is a novel non-invasive method to quantitate the brain metabolites besides Phe concentration in HPA patients. And it could be acquired conveniently on clinical MRI routine scanners. This study aimed to investigate the correlation between blood Phe ([Phe](blood)) and [Phe](brain), the characteristics of blood-brain Phe metabolism and its impacts on mental retardation.
METHODTotally 32 untreated patients diagnosed with HPA were studied, including 18 boys and 14 girls (age ranging from 33 days to 13 years). The patients were divided into two groups: elder than 4 months old (n = 22) and younger than 4 months old (n = 10). (1)HMRS were performed in all patients. [Phe](brain) were measured by absolute [Phe](brain) using Creatinine as an internal reference. [Phe](blood) were measured and developmental quotient (DQ) or intelligence quotients (IQ) were evaluated.
RESULT(1) [Phe](brain) measured by (1)HMRS ranged from 0.0640 to 0.6296 (M = 0.1542) while the [Phe](blood) was from 0.3804 to 2.5140 mmol/L (M = 1.5210 mmol/L) in all the 32 cases of HPA patients. (2) There was a positive linear correlation (r = 0.6103 (P < 0.01)) between [Phe](blood) and [Phe](brain). And there were interindividual differences in [Phe](brain) in several patients. (3) Variable mental retardation were observed in 23/32 cases in this study. (4) There was a negative correlation between [Phe](blood) and [Phe](brain) to the mental retardation (r(blood) = -0.5045, r(brain) = -0.6471 (P < 0.01)) in 22 cases of the HPA patients older than 4 months. And [Phe](brain) had more significant correlation with mental development than [Phe](blood).
CONCLUSIONThe [Phe](blood) could correspondingly represent the [Phe](brain) in most HPA patients. The Phe concentration could reflect the degree of mental retardation substantially in 22 cases with HPA older than 4 months. And the [Phe](brain) could more accurately illustrate it. (1)HMRS can be used to quantitate intracerebral Phe concentrations non-invasively in HPA patients. Preliminary findings suggest that interindividual variations in the kinetics of Phe uptake and metabolism do exist. (1)HMRS has great clinical significance in understanding the mechanism of HPA patient's mental retardation, providing proper objective standards for better diagnosis and treatment of HPA patients.
Adolescent ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Intelligence Tests ; Magnetic Resonance Spectroscopy ; Male ; Phenylalanine ; analysis ; Phenylketonurias ; metabolism
10.A new monacolin analogue from Xuezhikang capsule.
Xue-mei LI ; Xing-hai SHEN ; Zhen-wen DUAN ; Shu-ren GUO
Acta Pharmaceutica Sinica 2011;46(5):564-567
Xuezhikang capsule (ethanol extract of red yeast rice) which produced by Beijing WBL Peking University Biotech Co., Ltd., is a traditional Chinese medication with 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibiting activity. Clinical trials indicated that Xuezhikang with lipid-lowering action could reduce the risk of cardiac events and total mortality of Chinese coronary heart disease patients. To exactly explain the clinical features of Xuezhikang, we undertook a complete study of the chemical constituents of Xuezhikang. This study resulted in the isolation of a new monacolin analogue, named alpha, beta-dehydromonacolin L (1), along with two known compounds: monacolin L (2) and 3-(2, 6-dimethyl-1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalen-1-yl)propanoic acid (3). The chemical structures were determined by extensive 1D and 2D NMR and MS spectroscopic analysis.
Drugs, Chinese Herbal
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chemistry
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Molecular Structure
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Naphthalenes
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chemistry
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isolation & purification
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Propionates
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chemistry
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isolation & purification