Biomedical subjects

Dong-Zhi Wei

Publications and source records attributed to Dong-Zhi Wei.

At least 19 recordsLinked to original sources

PubMed2007

Some characteristics and purification of anti-(human ovarian carcinoma)xanti-(human CD3) single-chain bispecific antibody.

To obtain high-quality AhOC x AhCD3 [anti-(human ovarian carcinoma)xanti-(human CD3)] with high biological activity economically and easily, some characteristics and purification of AhOC x AhCD3, a single chain bispecific antibody, were investigated. During the present study, some important properties of AhOC x AhCD3, such as molecular mass, pI, solubility, stability, hydrophobic ability, molecular status and bioactivity were primarily studied. The molecular mass of AhOC x AhCD3 was determined to be approx. 58.0 kDa by SDS/PAGE (theoretical molecular mass: 56972.5 Da, calculated on the basis of the primary structure) and the pI was approx. 7.5+/-0.4 by IEF (isoelectric focusing; theoretical pI: 8.5, predicted by the ProtParam software tool) respectively. Experiments showed that the solubility of AhOC x AhCD3 increased with the pH increase and that the AhOC x AhCD3 was easily degraded into several fragments during the storage time of samples and the mixtures of both fragment B and fragment C retained normal bioactivity. Particularly, the soluble aggregate/polymer status of AhOC x AhCD3 with bioactivity was verified by non-reducing SDS/PAGE. After optimization of purification process, AhOC x AhCD3 with electrophoretic homogeneity was successfully obtained and the yield was approx. 20%. Some important properties of AhOC x AhCD3 were first observed and a procedure for soluble AhOC x AhCD3 purification in scale-up was first established. Some characteristics of AhOC x AhCD3, especially the stability of AhOC x AhCD3, are critical for the development of this protein pharmaceutical and useful for reference to other proteins.

Antibodies, Bispecific↗

PubMed2006

Penicillin acylase catalysis in the presence of ionic liquids.

Several ionic liquids were used as reaction media for penicillin G acylase catalysis. In all the assayed ionic liquids, [bmim]PF6 proved good media for PGA-catalyzed hydrolysis. A novel [bmim]PF6/water two-phase system is provided for 6-aminopenicillanic acid (APA) production, which will be more benefical than aquous batch systems used widely in industrial production of APA.

Catalysis↗

PubMed2006

Ganoderic acid T from Ganoderma lucidum mycelia induces mitochondria mediated apoptosis in lung cancer cells.

Ganoderma lucidum is a well-known traditional Chinese medicinal herb containing many bioactive compounds. Ganoderic acid T (GA-T), which is a lanostane triterpenoid purified from methanol extract of G. lucidum mycelia, was found to exert cytotoxicity on various human carcinoma cell lines in a dose-dependent manner, while it was less toxic to normal human cell lines. Animal experiments in vivo also showed that GA-T suppressed the growth of human solid tumor in athymic mice. It markedly inhibited the proliferation of a highly metastatic lung cancer cell line (95-D) by apoptosis induction and cell cycle arrest at G(1) phase. Moreover, reduction of mitochondria membrane potential (Delta psi(m)) and release of cytochrome c were observed during the induced apoptosis. Our data further indicate that the expression of proteins p53 and Bax in 95-D cells was increased in a time-dependent manner, whereas the expression of Bcl-2 was not significantly changed; thus the ratio of Bcl-2/Bax was decreased. The results show that the apoptosis induction of GA-T was mediated by mitochondrial dysfunctions. Furthermore, stimulation of the activity of caspase-3 but not caspase-8 was observed during apoptosis. The experiments using inhibitors of caspases (Z-VAD-FMK, Z-DEVD-FMK and Z-IETD-FMK) confirmed that caspase-3 was involved in the apoptosis. All our findings demonstrate that GA-T induced apoptosis of metastatic lung tumor cells through intrinsic pathway related to mitochondrial dysfunction and p53 expression, and it may be a potentially useful chemotherapeutic agent.

Animals↗

PubMed2006

Improvement of expression level and bioactivity of soluble tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) by a novel zinc ion feeding strategy.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) is a new member of the TNF superfamily. In this work, the key role of Zn(2+) in high-level expression of soluble TRAIL was confirmed. The yield of soluble TRAIL reached 1.6 g l(-1) using a novel, two-stage Zn(2+) feeding strategy, and the accumulation of TRAIL inclusion bodies decreased. Furthermore, the purified TRAIL showed stronger cytotoxicity activity against human pancreatic 1990 tumor cells as the molar ratio of Zn(2+) to TRAIL monomer was 2 in purified TRAIL solution.

Apoptosis↗

PubMed2006

In vitro anti-tumor activity of isorhamnetin isolated from Hippophae rhamnoides L. against BEL-7402 cells.

Isorhamnetin, a flavonol aglycone, isolated from the traditional Chinese medicine Hippophae rhamnoides L., was investigated in its cytotoxicity and its influence on human hepatocellular carcinoma cells (BEL-7402). The cytotoxic effects of isorhamnetin showed dose- and time-dependency against BEL-7402 cells, with IC(50) equal to 74.4+/-1.13 microg ml(-1) after treatment with isorhamnetin for 72 h. Cytotoxicity of the flavonols on tumor cells depends on cellular accumulation of the drugs. The amount of isorhamnetin accumulated in BEL-7402 cells was assayed by high-performance liquid chromatography (HPLC) and showed that isorhamnetin could permeate the cell membrane into the cell. Staining with Hoechst 33258 showed fragmentation and condensation of chromatin in the cell treated with 50 microg ml(-1) isorhamnetin for 48 h. Flow cytometric analysis was performed to determine hypodiploid cells. The results of flow cytometry assay indicated that the percentage of hypodiploid BEL-7402 cells were 13.77+/-1.05% after 48 h treatment with 50 microg ml(-1) isorhamnetin. The treatment resulted in the appearance of a hypodiploid peak (sub-G(0)/G(1) peak), probably due to the presence of cells in apoptosis and apoptotic bodies with DNA content less than 2n. To our knowledge, this is the first report against human hepatocellular carcinoma cells (BEL-7402) of isorhamnetin.

Antineoplastic Agents, Phytogenic↗

PubMed2006

Functional solubilization of aggregation-prone TRAIL protein facilitated by coexpressing with protein isoaspartate methyltranferase.

TRAIL was a tumor-specific protein in development as a novel anticancer therapeutic agent. Generally, when expressed in recombinant Escherichia coli, TRAIL protein was prone to form inclusion bodies. In this study, coexpression of human TRAIL protein and protein isoaspartate methyltranferase (PIMT) from E. coli on plasmid pBV-TRAIL-PCM in E. coli C600 was investigated to overcome the difficulties in soluble expression. The results showed that this PIMT coexpression strategy exerted a positive effect on the TRAIL protein expression in recombinant E. coli, which led to a mean increase in the intracellular concentration of soluble and total protein of TRAIL by 1.57-fold and 1.33-fold, respectively. At the same time, results also suggested that PIMT was a prospective partner for soluble expression of TRAIL protein.

Cloning, Molecular↗

PubMed2006

Partial purification of human parathyroid hormone 1-84 as a thioredoxin fusion form in recombinant Escherichia coli by thermoosmotic shock.

A modified purification method, thermoosmotic shock (osmotic shock coupled with heat-treatment) for heat-stable proteins, was devised in the purification of Trx-hPTH (1-84) (human parathyroid hormone coupled with thioredoxin as a fusion partner) from E. coli. Thermoosmotic shock can integrate the functions of extraction and crude separation of fusion protein Trx-hPTH (1-84). To improve the purification efficiency, thermoosmotic shock conditions were optimized and achieved as follows: the optimized high osmotic solution containing 20mM Tris-HCl buffer (pH 8.0), 1mM EDTA, and 25% sucrose; the low osmotic solution containing 20mM Tris-HCl buffer (pH 8.0), 1mM EDTA, and the heat-treatment temperature of 100 degrees C for 10 min. Using this method, the purity of Trx-hPTH (1-84) was up to 73% and the yield was up to 72%, respectively. In addition, the two separation methods of both thermoosmotic shock and affinity chromatography have been compared, indicating that thermoosmotic shock is an economical and feasible way for the fusion protein separation. Besides, the thermoosmotic shock method may be used for the purification of some proteins of thermal stability without N-terminal His-tag.

Chromatography, Affinity↗

PubMed2006

Determination of the amino acid sequence in a cyclic lipopeptide using MS with DHT mechanism.

A TOF MS/MS method to directly determine the amino acid sequence in a cyclic lipopeptide without its hydrolysis is described. The fragments of the peptide and the hydrocarbon chains were identified through comparing the MS of two analogues of the lipopeptide; the connecting relationship of amino acid residues in the lipopeptide was determined based on the difference of mass to charge ratio between peaks in the MS spectra and the amino acid analysis; and finally, according to the mechanism of double hydrogen transfer(DHT) the C-terminal of peptide and hydroxy aliphatic acid in the lipopeptide was directly determined without the hydrolysis. The determined sequence of amino acid residues in the cyclic lipopeptide is also supported by the rest peaks in the MS spectra grounded on simple fragmenting mechanism. This method can be used to determine the amino acid sequence in any aliphatic acid loop-inlaying cyclic lipopeptides.

Amino Acid Sequence↗

PubMed2006

Chromosomal integration of the Vitreoscilla hemoglobin gene and its physiological actions in Tremella fuciformis.

The Vitreoscilla hemoglobin (VHb) gene was expressed in yeast-like conidia (YLCs) of Tremella fuciformis (T. fuciformis) to increase cell density in submerged fermentation by enhancing oxygen uptake. With the intention of doing this, an integrated expression vector containing the VHb gene and the hygromycin B phosphotransferase (hph) gene derived from Escherichia coli (E. coli) as the selectable marker was constructed, and then transformed into protoplasts of YLCs from T. fuciformis with restriction enzyme-mediated DNA integration (REMI). Hygromycin-resistant transformants had been generated during the transformation. Molecular evidences including PCR assay, Southern blotting, and Western blot analysis indicated the VHb gene had been integrated into the genome of transgenic T. fuciformis strains and was expressed successfully. Shake-flask fermentation and bioreactor cultivation results showed that the expression of VHb in this fungus could enhance growth of YLCs. The final cell density was higher in the culture of VHb-expressing strain than that of the wild-type strain. Moreover, these results also suggested that CaMV35S promoter was capable of driving the expression of heterologous genes in T. fuciformis.

Bacterial Proteins↗

PubMed2006

Study on improving the selectivity of compounds that inhibit two PI3Ks (gamma and delta).

A desirable characteristic of PI3K inhibitors is their selectivity. Up to now, there has been no report that describes the 3 D-structure differences between two PI3Ks (delta and gamma) and applies them to designing selective compounds. In the present study, we used an approach combining protein-structure modeling, GRID/PCA (Principal Component Analysis) and docking methods to investigate the detail interactions of the two PI3Ks with various chemical groups. At first, we constructed a 3 D-model of the PI3Kdelta catalytic subunit with the program Modeller7.0 based on the high resolution X-ray structure of the PI3Kgamma catalytic subunit, and then employed GRID and PCA to reveal the most relevant structural and physicochemical differences between the two PI3Ks related to their selectivity. As a result, the analysis unveiled the most important regions on the two PI3Ks that should be taken into account for the design of selective inhibitors. Finally, based on activity data of 10 PI3Kdelta-selective compounds, a docking study validated the results of the GRID/PCA method, which suggested that the approach could provide clear guidelines for selective drug design.

Amino Acid Sequence↗

PubMed2006

[Preparation of recombinant HIV-TATm-survivin (T34A) protein and its pro-apoptosis activity to cancer cells in vitro].

As a novel member of the IAP (Inhibitor of apoptosis protein) family, survivin was observed to be expressed in most human cancerous cells. Fusion protein TATm-survivin (T34A) has drawn considerable attention because it is a potential anti-tumor protein that can be transduced into cancer cell with the help of HIV-TAT domain. In this study, the cDNA encoding survivin was cloned by RT-PCR from human breast cancer cell lines B-Cap-37. An expression vector of pRSET-B-HIV-tatm-survivin (T34A) was constructed by PCR after survivin (T34A) was mutated by site-directed mutagenesis. Subsequently, the resultant plasmid was transformed into E. coli BL21 (DE3). Recombinant HIV-TATm-Survivin (T34A) protein was expressed efficiently with 0.5mM IPTG as inducer, reaching a yield of 650mg/liter (as inclusion body) in fermentation culture. The inclusion bodies were solubilized, refolded and purified to a purity of 96% by ion exchange chromatograghy and size-exclusion chromatography. Remarkable effects of the purified recombinant HIV-TATm-Survivin (T34A) on the morphology of cell line SW1990 and B-Cap-37 were observed after being administrated for 4h. MTT assay showed recombinant HIV-TATm-survivin (T34A) protein could inhibit significantly cell proliferation of SW1990 and B-Cap-37 and SSMC-7721 in vitro. Apoptosis rate and cell circle of SW1990 and B-Cap-37 that had been treated with target protein (final concentration 30 microg/mL) were detected with flow cytometry. Results revealed that more than 65% cancer cells were arrested at G1 phase. The study suggested that TATm-survivin (T34A) protein was a hopeful protein drug in the treatment of cancers by facilitating apoptosis of cancer cells. Key words recombinant HIV-TATm-Survivin (T34A), expression and purification, pro-apoptosis bioactivity, SW1990 and B-Cap-37 cancer cell lines

Apoptosis↗

PubMed2006

Preparation of optically pure tert-leucine by penicillin G acylase-catalyzed resolution.

Penicillin G acylase, from Kluyvera citrophila, was used in kinetic resolution of DL-tert-leucine. N-phenylacetylated-DL-tert-leucine, chemically synthesized from DL-tert-leucine, was enantioselctively hydrolyzed by penicillin G acylase to obtain L-tert-leucine, D-tert-leucine was prepared by acid-catalyzed hydrolysis of the remaining substrate. The total yields of D-tert-leucine and L-tert-leucine are 80.6% and 83.1%, respectively. The enantiomeric excess of the two products, D-tert-leucine and L-tert-leucine, are 98.5% and 99%. This is a practical way for the preparation of D-tert-leucine and L-tert-leucine.

Catalysis↗

PubMed2006

Mutant library construction in directed molecular evolution: casting a wider net.

Directed molecular evolution imitates the natural selection process in the laboratory to find mutant proteins with improved properties in the expected aspects by exploring the encoding sequence space. The success of directed molecular evolution experiment depends on the quality of artificially prepared mutant libraries and the availability of convenient high-throughput screening methods. Well-prepared libraries promise the possibility of obtaining desired mutants by screening a library containing a relatively small number of mutants. This article summarizes and reviews the currently available methodologies widely used in directed evolution practices in the hope of providing a general reference for library construction. These methods include error-prone polymerase chain reaction (epPCR), oligonucleotide-based mutagenesis, and genetic recombination exemplified by DNA shuffling and its derivatives. Another designed method is also discussed, in which B-lymphocytes are fooled to mutate nonantibody foreign proteins through somatic hypermutation (SHM).

Animals↗

PubMed2005

Effect of organic cosolvent on kinetic resolution of tert-leucine by penicillin G acylase from Kluyvera citrophila.

Penicillin G acylase (PGA) from Kluyvera citrophila immobilized on Amberzyml was used for enantioselective hydrolysis of N-phenylacetylated-DL-tert-leucine (N-Phac-DL-Tle) to produce L-tert-leucine (L-Tle). The effects of various organic cosolvents on hydrolysis of N-Phac-DL-Tle have been investigated in aqueous-cosolvent medium. It was founded that the rate of PGA-catalyzed reaction was significantly affected by the presence of 2% (v/v) organic cosolvent concentration. The initial rate fell with increasing logP of the cosolvent, but for logP values less than -0.24 the rate was faster than in purely aqueous medium. Additionally, the relative rate increases with the increase of dielectric constant (epsilon) of organic cosolvents. The yields of L-Tle in all aqueous-cosolvent systems were above 95% with the enantiomeric excess (ee) of >99%.

Enzyme Activation↗

PubMed2005

Optimization of culture on the overproduction of TRAIL in high-cell-density culture by recombinant Escherichia coli.

Different nutrient-feeding cultures were carried out in producing recombinant protein of truncated tumor necrosis factor related apoptosis-inducing ligand (TRAIL) (114-281 amino acids of TRAIL) in Escherichia coli strain C600/pBV-TRAIL. The effects of preinduction specific growth rate, postinduction carbon source (glucose and glycerol), and feeding strategies were investigated. The higher preinduction specific growth rate (mu = 0.22 h(-1)) contributed to the increase in the TRAIL production, at which TRAIL was accumulated in bacterial cells as 7.2% of total cellular protein, corresponding to 1.99 g l(-1) in contrast with 5.1% (1.29 g l(-1)) at preinduction specific growth rate (mu = 0.1 h(-1)) during high-cell-density culture. Glycerol was superior to glucose as the postinduction carbon source for TRAIL production. Under similar culture conditions, the final concentration of TRAIL was produced 1.59-fold more when glycerol was used as postinduction carbon source than when glucose was used. At the same time, the results showed that it is efficient to adopt the pH-stat feeding strategy at postinduction for the overproduction of TRAIL. The TRAIL production was increased up to 4.51 g l(-1), approximately 16.1% of total cellular protein. The mechanisms behind the preinduction specific growth rate effect on the expression level may be ascribed to the leakage secretion of acetate.

Acetates↗

PubMed2005

Nuclear localization signal of ING4 plays a key role in its binding to p53.

ING4, a novel member of ING family, is recently reported to interact with tumor suppressor p53 and negatively regulate the cell growth with significant G2/M arrest of cell cycle in HepG2 cells through upregulation of p53-inducible gene p21. However, which region of ING4 could have contributed to the binding to p53 remains largely unclear. Herein, the GST-pulldown experiments revealed that the middle region of ING4, a potential bipartite nuclear localization signal (NLS), could be involved in the binding to p53. Furthermore, the interaction of ING4 to p53 was abrogated in vitro and in vivo when certain mutations or the entire deletion of the NLS domain occurred. More interestingly, the mutations of the NLS domain could alter the ING4 nuclear localization, disrupt the interaction of ING4 with p53, and even, deregulate the p53-inducible gene p21 in MCF-7 cells. All data indicated that the NLS domain of ING4 is essential for the binding of ING4 to p53 and the function of ING4 associated with p53.

Amino Acid Sequence↗

PubMed2005

In vivo antitumor activity by 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone in a solid human carcinoma xenograft model.

Previously we have shown that 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), which is isolated from the buds of Cleistocalyx operculatus, significantly inhibits the growth of human liver cancer SMMC-7721 cells and is able to induce apoptosis of SMMC-7721 cells in vitro. Here we report the antitumor effects of DMC in vivo, using a solid human tumor xenograft mouse model using human liver cancer SMMC-7721 cells. The average tumor weights in the control group and in mice injected with 150 mg/kg DMC were 1.42+/-0.11 g and 0.59+/-0.12 g, respectively. Flow cytometric analysis of the tumor cell population demonstrated an aneuploid peak (representing 33.60+/-0.80% of the total in mice injected with 150 mg/kg DMC). To our knowledge, this is the first time that chalcone compounds have been applied to a human tumor xenograft model.

Animals↗

PubMed2005

Induction of apoptosis in K562 human leukemia cells by 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone.

2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), isolated from the buds of Cleistocalyx operculatus, was investigated in its cytotoxicity and anti-proliferation on K562 cell line. Our results revealed that the IC50 was equal to 14.2+/-0.45 microM and the EC50 was 3.3+/-0.14 microM. Staining with Hoechst 33258 showed fragmentation and condensation of chromatin in the cells treated with 8 microM DMC for 48 h. Flow cytometric analysis was performed to determine hypodiploid cells. The results of flow cytometry assay indicated that the percentage of hypodiploid K562 cells was 76.15+/-3.22% after 48 h treatment with 16.0 microM DMC. The treatment resulted in the appearance of a hypodiploid peak (A0 region), probably due to the presence of apoptosing cells and/or apoptotic bodies with DNA content less than 2n. Western blot results illustrated that in the same dosage and incubation time, DMC could down-regulate the level of Bcl-2 protein and did not influence the expression of Bax protein. The resulting net effect could thus lead to a lower ratio of Bcl-2/Bax, which might be responsible for the DMC-induced apoptosis in K562 cells.

Apoptosis↗