Biomedical subjects
Publications and source records attributed to S L Yang.
A snake venom gene TSV-PA was inserted into the donor plasmids pFastBacHTa and expressed in Tn-5B1-4 cells. SDS-PAGE analysis revealed that the molecular weight of expressed product of TSV-PA were 33 kD. It was also proved by Western blot. The result of enzyme activity showed that TSV-PA protein expressed in insect cells had a higher activity.
We have identified the bottleneck steps limiting maturation of penicillin G acylase (PAC) through comparison of the maturation performance for various PAC-expression systems (Pac, Tac, T7, Vgb + T7) with different efficiencies of proteolysis, subunit folding and assembly. The maturation of PAC could be limited by various steps, such as translocation, periplasmic proteolysis, subunit folding and assembly depending on the host/vector systems. In BL21(pPA6) cells, maturation of PAC were limited by proteolysis and folding steps; the efficiency of proteolysis was 57.2%; the subunit folding and assembly capacity was 0.72. In BL21(pKKpacSP) cells, the stability and folding of alpha subunit was bottleneck steps. In T7 and dissolved-oxygen regulation expression systems, PAC proprecursor could be maturated efficiently. Results also indicate that the folding of alpha peptide plays a key role in folding of precursor for PAC in E. coli. Developing proper host/vector systems and fermentation technology with superior abilities on subunit folding and assembly of precursor for PAC could be plausible for enhancing production of PAC. In this study, pac could be expressed (transcribed, translated and maturated) efficiently under the control of T7 promoter.
OBJECTIVE: To investigate the organic acid constituents from the stem of Securidaca inappendiculata. METHOD: Column chromatographic techniques were used to isolate and purify the constituents. The structures were elucidated by spectra analysis. RESULT: Five compounds were isolated and identified as ferulic acid (I), cinnamic acid(II), palmitic acid(III), salicylic acid(IV) and benzoic acid(V). CONCLUSION: Compounds I-IV were isolated from the plant for the first time.
OBJECTIVE: To study the chemical constituents in the volatile oil from fruits of Alpinia oxyphylla. METHOD: Using GC-MS to identify the constituents. RESULT AND CONCLUSION: Sixty-four compounds were identified on the basis of GC-MS, the main ones being p-cymene, valence, linalool, myrtenal, alpha-pinene, beta-pinene, furopelargone A and terpinen-4-ol. Three sesquiterpenes valencene, nootkanone and nootkanol have been isolated from the CHCl3 extract as check, of these 64 identified compounds linalyl oxide, valencene, bakkenolide A, furopelargone A and 3-hydroxycalamenene are reported for the first time.
OBJECTIVE: To study the chemical constituents the roots of Chirita fimbrisepala. METHOD: The constituents were extracted with solvent, separated and purified with chromatographic methods, identified by NMR, MS, UV, IR and physical-chemical constants. RESULT: Three flavonoids mahuangchiside(I), hispidulin (II) and kaempferol(III) were isolated with daucosteral(IV). CONCLUSION: I is a new compound elucidated as hispidulin-7-O-beta-D-xylopyranosyl-(1-->2)-beta-D-xylopyranoside, named mahuangchiside, II and III were isolated for the first time from the family Gesneriaceae, and IV was isolated for the first time from the genus Chirita.
OBJECTIVE: To study the chemical components in the stem of Dasymaschalon rostratum. METHOD: The components were extracted with solvent, separated and purified with chromatographic methods, identified by NMR, MS, UV, IR and physicol-chemical constants. RESULTS: Three A-ring-formylated flavonoids and one oxoaporphinnoid aikaloid were isolated and identified as lawinal, unonal, isounonal and 7-oxodehydroasimilobine. CONCLUSION: All the four compounds were isolated for the first time from the genus Dasymaschalon. According to all the phytochemistry papers on Annonaceae, A-ring formylated flavonoids in this family were isolated from the genus Desmos for the first time. Thus, it is an interesting discovery in chemotaxonomy which reveals the close relationship between the two genera Desmos and Dasymaschalon.
OBJECTIVE: To study the chemical constituents in the fruit of Lycium barbarum. METHOD: The chemical constituents were isolated by column chromatography and identified by spectral data. RESULT: The compounds obtained were identified as scopoletin(I), beta-sitosterol(II), p-coumaric acid(III), glucose(IV), daucosterol(V) and betaine(VI). CONCLUSION: Compounds III, IV and V were isolated from Lycium barbarum for the first time.
AIM: To develop a method for analysis of antitumor annonaceous acetogenins in Annonaceae plants by HPLC. METHODS: Squamostatin-B (1), squamocin (2) and annonin-VI (3) were used as standard substances. Chromatography column was a Rp-18; the mobile phase was methanol-water (90:10); the flow rate was 1.0 mL.min-1 and the detecting wavelength was 220 nm. RESULTS: A linear range was obtained from 2.3 to 13.8 micrograms with a good correlation. The recoveries of (1), (2) and (3) were 100.3%, 100.3% and 100.0%, respectively. CONCLUSION: This method was developed for the analysis of acetogenins by HPLC for the first time. The method is rapid, accurate and suitable for the analysis of the antitumor acetogenins in Annonaceae plants.
To eliminate the harm of mercury, we, by means of special technology, have developed a new type of dental filling material, comprising of glass ionomer and 100% ultra-fine ball-shaped silver amalgam powder. Clinical applications show it has very good wear-resistant, pressure-resistant and adhesive properties Moreover, it causes no irritation to the dental pulp and is a good kind of dental filling material in clinical applications.
OBJECTIVE: To compare the economical characters, yield characters and content of catalpol on Rehmannia glutinosa Libosch. varieties. METHODS: To study characters by field randomized block test and analysis of variance. To analyse the content of catalpol by HPLC. RESULTS: The results by analysis of variance were that the F value in plant width was 15.4 (F0.01 = 5.54), the F value in length of leaves was 12.2, the F value in width of leaves was 13.35, the F value in yield of single plant was 55.7 and the F value in content of catalpol was 8.03. The results by correlative analysis were that the linear correlation coefficient of signal plant yield with length of leaves was 0.9639, with width of leaves was 0.9073, with amount of earthnuts was 0.7060 and with plant fresh weight was 0.9950. The linear correlation coefficient of content of catalpol with plant width was 0.9169, with length of leaves was 0.7046 and with width of leaves was 0.7159. CONCLUSION: There were significant differences in plant width, length of leaves width of leaves, and yield of single plant and content of catalpol of Rehmannia glutinosa Libnosch. varieties. There were significantly positive correlations in signal plant yield with plant fresh weight, length of leaves, width of leaves and amount of earthnuts. There were significantly positive correlations in content of catalpol with plant width, length of leaves and width of leaves.
By using intrasplenic immunization and the conventional B lymphocyte hybridoma technique, we have established two novel hybridoma cell lines stably secreting specific monoclonal antibodies (MAbs) to magaininII, termed as 2D1 and 3F8, respectively. The two cell lines were then subjected to RNA extraction and the VH and VL segments were obtained by reverse transcription of RNA followed by polymerase chain reaction (RT-PCR) and characterized by nucleotide sequence analysis. The VH segments of 2D1 and 3F8 belong to the VH5 family and the VL segments of 2D1 and 3F8 belong to VK10 and VK1 groups, respectively. The two MAbs utilize different VL segments and have disparities in their HCDR3 regions, which may contribute to the different epitope recognition of the two antibodies.
Klebsiella pneumoniae is a common cause of septicemia and urinary tract infections. The PCR-supported genomic subtractive hybridization was employed to identify genes specifically present in a virulent strain of K. pneumoniae. Analysis of 25 subtracted DNA clones has revealed 19 distinct nucleotide sequences. Two of the sequences were found to be the genes encoding the transposase of Tn3926 and a capsule polysaccharide exporting enzyme. Three sequences displayed moderate homology with bvgAS, which encodes a two-component signal transduction system in Bordetella pertussis. The rest of the sequences did not exhibit homology with any known genes. The distribution of these novel sequences varied greatly in K. pneumoniae clinical isolates, reflecting the heterogeneous nature of the K. pneumoniae population.
Bu-Zhong-Yi-Qi-Tang (BZYQT) is a Chinese medicine, and has been used for the treatment of hepatocellular carcinoma (HCC) patients. At present, we still do not fully understand the effects of BZYQT on the cellular physiology. Present in vitro study demonstrated that BZYQT is capable of increasing granulocyte colony-stimulating-factor (G-CSF) and tumor necrosis factor-alpha (TNF-alpha) production by peripheral blood mononuclear cells (PBMC) in healthy volunteers and patients with HCC. The productions of G-CSF and TNF-alpha by PBMC of volunteers were significantly stimulated by more than 125 microg/ml of BZYQT. G-CSF levels stimulated by PBMC of healthy volunteers were higher than in PBMC of the HCC patients when more than 625 microg/ml of BZYQT was administrated. The reason may be due to the impaired immunologic reactivity of mononuclear cells in HCC patients. However, the production levels of TNF-alpha in HCC patients can be stimulated to levels as high as those in healthy volunteers. When adding high concentration (3.125 mg/ml) of BZYQT to the cultured PBMC, the increments of G-CSF and TNF-alpha production decreased although there were no obvious changes in the number of metabolic active PBMC changed. TNF-alpha andG-CSF are known to play important roles in the biological defensive mechanism. These findings show that BZYQT is a unique formula for the stimulation of PBMC to produce G-CSF and TNF-alpha. Administration of BZYQT may be beneficial for patients with HCC to modulate these cytokines.
Ser 236, located in the surface of subtilisin E and at the end of alpha-helex with Ser221, is far from the active center (Asp32, His64, Ser221). The replacement of Ser236 with Cys via directed-mutagenesis should effect protease activity insignificantly and may be able to improve the stability of enzyme due to the potency to form disulfide across two molecules. The results indicated that BP-1 (Ser236Cys) was found to gain 150% activity (Kcat/Km value) of the wild-type subtilisin E and 3-fold increase in stability in aqueous solution at pH7.4 and 50 degrees C, compared to the wild-type enzyme. However, further mutations on BP-1 to introduce Ala15Asp/Gly20His (BU-1) or Ser24His/Lys27Asp (BW-1) was found negative effects on the activity and stability of subtilisin E.
The possibility of using lactose as an inducer to substitute the common inducer IPTG in the fermentation process of the recombinant microorganism was deeply investigated. The influences of culture conditions such as lactose concentration, growth medium composition, the point of induction and the duration of the induction phase on the expression of the recombinant protein were analyzed and studied in detail. In the following experiments, lactose was then used in the high cell density culture process of E. coli BL21 (DE3)(pFu). The final cell density (OD600) was over 40. The expression level of recombinant protein was about 15% of the total cellular protein. Both the culture density and foreign protein expression level were lower than those induced by IPTG. However, because of the potential toxicity to human beings and the high cost of IPTG, the use of lactose might provide an alternative means of inducing foreign protein expression. This would be more attractive in industrial scale productions of recombinant proteins. The results confirmed that lactose could be used as an inducer in the fermentation process.
The cDNA encoding the precursor of cobrotoxin was cloned from the venom gland of the Chinese continental cobra (Naja naja atra) by RT-PCR. Its deduced amino acid sequence analysis showed that the mature protein was identical to that identified from the Taiwan cobra (Naja naja atra) by protein sequencing technique. The cDNA encoding the mature protein was then subcloned into the expression vector pMAL-P2. The gene of CM11, which was formed by ligation of the fragments of the synthetic oligonucleotides, was also cloned into the expression vector pMAL-P2. After induction of IPTG, both of the neurotoxins were overexpressed as soluble fusion proteins which were confirmed by SDS-PAGE and western blotting. The expressed fusion proteins was purified by sepharose 6B-amylose affinity chromatography and DEAE-sepharose FF chromatography. Both of the recombinant proteins achieved after digestion by factor Xa showed the in vivo toxicity.
A human granulocyte-macrophage colony stimulating factor (GM-CSF)/interleukin-3(IL-3) fusion gene with a short linker between the GM-CSF and IL-3 gene has been successfully constructed and expressed in E. coli under the control of T7 promoter. The recombinant fusion protein was expressed as inclusion bodies after the IPTG induction. The yield of the GM-CSF/IL-3 fusion protein was over 30% of the total cellular proteins. Western-blotting results showed that the fusion protein could specifically combined with GM-CSF antibody and IL-3 antibody. The biological activity was detected by the GM-CSF and IL-3 dependent cell line TF-1. After solubilizing with 8 mol/L urea and renaturing with dialysis against Tris. HCl solution, the refolded fusion protein showed obvious activities to maintain the growth of TF-1 cell.
Site-directed mutagenesis was used to investigate the effects of S221C/P225A, N118S/S221C/P225A, D60N/N118S/S221C/P225A and Q103R/N118S/S221C/P225A mutations on the properties of Subtilisin E. It was found that S221C/P225A mutant is 73,000-fold decreased in amidase activity than subtilisin E and 3-fold increased than subtiligase in the ratio of esterase/amidase; N118S/S221C/P225A mutant has 3.6-fold and 15-fold decreased in amidase and esterase activity respectively and as a result, it has a 4-fold lower in the ratio of amidase/esterase than S221C/P225A mutant; Although it has no effect on the esterase activity, D60N/N118S/S221C/P225A mutant enhanced its ratio of amidase/esterase by 15 fold, 3.3-fold and 10.3 fold compared to N118S/S221C/P225A mutant, S221C/P225A mutant and subtiligase respectively; Q103R/N118S/S221C/P225A mutant, however, has a 5-fold enhanced in the amidase activity and 55-fold and 1000-fold decrease in the esterase activity and the ratio of esterase/amidase compared to N118S/S221C/P225A.