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Biomedical subjects

X F Wu

Publications and source records attributed to X F Wu.

At least 37 records · Page 2Linked to original sources

Cloning of a galactose-binding lectin from the venom of Trimeresurus stejnegeri.

A galactose-binding lectin isolated from the venom of Trimeresurus stejnegeri is a homodimer C-type lectin. The cloned cDNA encoding the monomer of Trimeresurus stejnegeri lectin (TSL) was sequenced and found to contain a 5'-end non-coding region, a sequence which encodes 135 amino acids, including a typical 23 amino acid signal peptide followed by the mature protein sequence, a 3'-end non-coding region, a polyadenylation signal, and a poly(A) region. To completely characterize the deduced amino acid sequence, on-line HPLC-MS and tandem MS were used to analyse the intact monomer and its proteolytic peptides. A modified peptide fragment was also putatively identified by HPLC-MS analysis. The deduced amino acid sequence was found to contain a carbohydrate-recognition domain homologous with those of some known C-type animal lectins. Thus TSL belongs to group VII of the C-type animal lectins as classified by Drickamer [(1993) Prog. Nucleic Acid Res. Mol. Biol. 45, 207-232]. At present, a number of C-type lectins have been purified from snake venom, but most of them have been characterized only at the protein level. To our knowledge, this is the first known cDNA sequence of a true C-type lectin from snake venom.

Amino Acid Sequence↗

The gene expression of coagulation factor VIII in mammalian cell lines.

Both the full-length and B domain-deleted cDNA of factor VIII were constructed in plasmid pcDNA3, respectively, and successfully expressed in Cos-7 cells. The yield of recombinant factor VIII-deltaB (0.4 U/mL/10(6) cells/day) was approximately four times higher than that of the recombinant factor VIII. In addition, it was indicated that the gene expression of factor VIII is specific for cells from different tissues. The highest expression level was found in the hepatocellular carcinoma line SMMC-7721, followed by kidney, ovary, and lung cell lines. To compare the efficiency of gene expression of recombinant factor VIII, the factor VIII-deltaB gene was further reconstructed in different forms in the expression plasmid pCMV-dhfr for transient gene expression in Chinese hamster ovary cells. The redundant 5'- and 3'-untranslated sequences of factor VIII-deltaB were deleted. The cDNA encoding the heavy and light chains of factor VIII were constructed, respectively. Among them the high yield of the recombinant factor VIII was found in the coexpression of the heavy and light chain cDNA fragments of factor VIII. The deletion of the redundant 5'-untranslated sequence of factor VIII-deltaB was also beneficial for gene expression. As expected, the gene coexpression of factor VIII-deltaB and von Willibrand Factor cloned by the long-polymerase chain reaction method was also helpful for enhancing the expression level of recombinant factor VIII. A monoclonal antibody raised against factor VIII was prepared and used for the specific assay of recombinant factor VIII by the competitive ELISA method, the assay results were consistent with those determined by the one-stage bioassay.

Animals↗

Cloning of a cDNA encoding a nerve growth factor precursor from the Agkistrodon halys Pallas.

Based on the high conservation in the 5' and 3' untranslated regions of NGF cDNAs, oligonucleotides complementary to all these known sequences were synthesized. By RT-PCR, we successfully isolated the complementary DNA encoding NGF precursor from the Agkistrodon halys Pallas (a Chinese snake strain). The nucleotide sequence which presents 90.5%, 88.6% and 63.4% homology to that of Krait Bungarus multicinctus, cobra and human NGF respectively, encoded a prepro-NGF molecule with 241 amino acids and a mature NGF molecule with 119 amino acids. The NGF cDNA inserts were subcloned into pCDNA3 expression vector and then transfected into COS-7 cells. The supernatant of the transfected cells turned out NGF biological activity as assayed by the survival rate of PC12 pheochromocytoma cells.

Agkistrodon↗

[Expression of chimeric single-chain antibody with specificity for HBsAg in E. coli].

The Vk gene of MAb with specificity for HBsAg and human Ck gene have been combined to form chimeric light chain (Vk-Ck) by recombinant PCR, which has combined with VH to construct chimeric single-chain antibody(ScFv-Ck) by a linker encoding a flexible peptide[(Gly3 Ser)3]. The ScFv-Ck has been expressed in the heat-induced expression system and secreted expression system of E. coli respectively. Analysis by Western-blot and indirect ELISA shows that the ScFv-Ck product in two systems both have HBsAg-binding ability, and ScFv-Ck has been secreted from E. coli in the direction of secretion peptide.

Blotting, Western↗

Diversity of cDNAs encoding phospholipase A2 from Agkistrodon halys pallas venom, and its expression in E. coli.

As a step toward understanding the structure and function of phospholipase A2(PLA2), we isolated several novel cDNAs encoding Agkistrodon halys Pallas PLA2 isoenzymes including B-PLA2, Asn49-PLA2, A-PLA2, A'-PLA2 and BA1-PLA2 by polymerase chain reaction with oligonucleotide primers corresponding to the N- and C-terminus of these enzymes. The amino acid sequences of A-PLA2 deduced from cDNA are consistent with that isolated from venom except for four residues. Asn49-PLA2 and B-PLA2 are highly similar (> 95%), but the critical residue Asp49 in the active centre of B-PLA2 is replaced by Asn49 in Asn49-PLA2. The N-terminal residues (1-24) of BA1-PLA2 shows high similarity to that of B-PLA2 which has strong ability to hemolyze erythrocytes, while its C-terminal residues (72-125) are the same as that of A-PLA2 which can inhibit platelet aggregation. The successful cloning of these isoenzymes not only provide excellent native material to study the structure-function relationship of PLA2s, but also to disclose the genesis of structural diversity of PLA2s, namely DNA modification and gene rearrangement. The cloned cDNA for A-PLA2 has been expressed in E. coli. By Q-Sepharose column chromatography, denaturation-renaturation and FPLC, we obtained the active recombinant protein with the initiator Met. This is the first report of the production of an active recombinant PLA2 with the initiator Met.

Agkistrodon↗

Preparation of highly purified hemoglobin by affinity elution.

The large scale production of recombinant hemoglobin (Hb) from microorganism or transgenic hosts for Hb-based blood substitutes places utmost emphasis on purity. In the present study, a high-resolution, convenient and inexpensive purification method is developed for purification of Hb from mixtures containing E. coli extract and bovine serum. This method is based on affinity elution by pyrophosphate (PPi) of Hb adsorbed on an FPLC column of the anion exchanger Toyopearl DEAE-650M. Compared to pH elution or NaCl elution, PPi elution makes possible the preparation of Hb of much higher purity. A procedure combining pH elution and PPi elution sequentially using a single column proves particularly valuable. The purification method is also applicable to the purification of cyanomet-Hb (CNHb+).

Chromatography, Affinity↗

Purification, cDNA cloning and molecular characteristic of a fibrinolytic enzyme from the venom of Agkistrodon acutus.

A nonglycoprotein-like fibrinolytic enzyme ((FIB-I) was purified from the crude venom of Agkistrodon acutus by CM-Sepharose CL-6B and DEAE-Sepharose CL-6B ion exchange chromatography and then by FPLC through Superose 12 gel filtration. Its molecular weight is about 23 kDa and isoelectric point is near 6.0. It not only has fibrinolytic and caseinolytic activity, but also can hydrolyze BAEE. The local hemorrhagic activity was found in mice after the subcutaneous injection of this enzyme. EDTA can inhibit its fibrinolytic activity completely, but PMSF and arrowhead proteinase inhibitor have no such obvious inhibitory effect, thus implying that FIB-I is a metalloproteinase. The N-terminal ten amino acid residues 'STEFQRYMEI' of FIB-I was elucidated. A full-length cDNA gene of this enzyme was cloned by using RT-PCR from the total RNA extracted from the snake venom gland and FIB-I was expressed in E. coli. Having analyzed the sequence, we found that it had a typical zinc-chelating characteristic as 'HEXXHXXGXXHD.'

Agkistrodon↗

Reconstruction and expression of chimeric anti-HBx antibody in Escherichia coli.

The variable regions of murine monoclonal anti-HBx immunoglobulin and the constant region of human antibody were cloned by reverse transcript-polymerase chain reaction (RT-PCR). The heavy-chain and light-chain variable regions were connected and coexpressed with human constant region C-r3 and C-k3 in the reconstructed vector of E. coli. The products showed high specificity and binding ability with HBx. Which is closely associated with hepatocarcinogenesis. This makes it possible to humanize the mouse monoclonal antibodies and express the fusion protein in E.coli for potential radioimmunotherapy in patients with hepatocellular carcinoma.

Animals↗

[Expression of human 17 beta-hydroxysteroid dehydrogenase gene in BmNPV expression system].

Estrogenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) plays a crucial role in the synthesis of estrogens, but it also produces negative action of estrogens in promoting the growth of hormone-sensitive cancers, especially in breast and prostate. The high specific activity can be taken as an important signal for the diagnosis of cancers. Recombinant rAcBm-NPV/17 beta-HSD virus which contains the human 17 beta-HSD cDNA under the control of polyhedron gene promoter is generated by cotransfection of the BmN cells with the transfer plasmid pVL/17 beta-HSD and wild BmNPV genomic DNA. 17 beta-HSD is maximally expressed 72 h and 120 h post infection in BmN cells and the 5th instar silkworm larvae respectively. At those time interval, intracellular and hemolymphic enzymatic activity reach 0.12 U/mg and 0.15 U/mg of protein which produced total activity of 0.97 U/1.5 x 10(6) cells and 4.7 U/larva. The expressed quantities in female larvae are a little higher than that in male larvae. The present data shows that Silkworm/BmNPV expression system can express 3-5 times higher than that of the richest human placenta. It also indicates that there is an apparent band with a molecular mass of 35 kDa using SDS-PAGE method, the size of which is similar to that of the crude enzyme from placenta.

17-Hydroxysteroid Dehydrogenases↗

mRNA levels of nm23 in murine ascites hepatoma (H22) clones with different lymphatic metastatic potential.

Levels of expression of the nm23 gene inversely correlated with metastatic potential in several rodent tumor model systems and human breast carcinoma. In the present study, we examined nm23 mRNA levels in two murine ascites hepatoma models (H22-16A3-F and H22-A2-P) with different metastatic potentials. Metastatic H22-16A3-F (80% metastatic rate) and non-metastatic H22-A2-P clones were both derived from murine ascites hepatoma (H22). We found that a 0.8-kb nm23 transcript was expressed in both cell clones. The nm23 gene was expressed at a higher level in non-metastatic H22-A2-P: approximately 8.6-fold higher than in metastatic H22-16A3-F. The present data suggest that the expression of nm23 mRNA might be associated with metastasis of murine ascites hepatoma (H22), though heterogeneity of nm23 steady-state expression levels among the H22 clones remains to be investigated.

Animals↗

An epidemiological study of risk factors for lung cancer in Guangzhou, China.

Lung cancer has been on a rapid rise worldwide during the last three or four decades, in part due to modern social habits and unhealthy lifestyles. Although smoking, air pollution, and certain types of occupational exposure have been recognized as the major risk factors for lung cancer, the significance of each of these factors appears to vary with sex, country, and with region within a given country. In the case of nonsmoking females, some risk factors for lung cancer remain to be identified. In the city of Guangzhou, lung cancer is one of the five leading tumors and the rate has been increasing steadily in both males and females since the 1970s. In this report, more than 6000 cases of lung cancer deaths, accumulated over the past 9 years, were analyzed. The severity of air pollution and cigarette smoking were positively correlated with the incidence of lung cancer deaths. Analysis of levels of SO2 and NOx suggests that the major source of indoor air pollution came from cooking. Two studies were performed in order to determine the relative contribution and importance of smoking, indoor air pollution and occupational exposure as risk factors for the rising incidence of lung cancer. The first was a population-based case-control study involving 849 subjects (566 males and 283 females). The second study was based on the data made available by the Third National Census survey, in which the standardized mortality rate (SMR) and population attributable risk (PAR) for lung cancer due to occupational exposure for the population in Guangzhou were analyzed. Results of these two studies show that: in females, indoor air pollution, derived primarily from burning coal, was found to be a highly significant risk factor for lung cancer. In males, however, cigarette smoking and occupational exposure were significantly associated with lung cancer. To further elucidate the contribution of indoor air pollution as a risk factor for lung cancer in nonsmoking females, two additional case-control studies were performed in 1985 and 1986. The 1985 study involved 120 nonsmokers (28 males, 92 females) in which the influence of such lifestyle factors as: personal history of nonmalignant respiratory diseases, fresh vegetable consumption, lifetime occupation and occupational exposure histories, exposure to environmental tobacco smoke (ETS), degree of indoor air pollution, general conditions of home residence, cooking practices and environments, and family history of cancer were first individually assessed and then collectively subjected to multiple conditional regression analysis for evaluation as risk factors for lung cancer. The 1986 study involved 75 cases of never-smoking females in which the aim was to investigate the influence of exposure to spousal smoke as a risk factor for lung cancer. These studies suggest that consumption of fresh vegetables was a "protective" factor for lung cancer in both males and females. In females, indoor air pollution and size of the kitchen were risk factors for lung cancer, whereas ETS exposure, respiratory disease history, family history of cancer, living conditions, use of cooking fuel, and participation in cooking, were not statistically associated with female lung cancer deaths. Occupational exposure was also correlated with the incidence of female lung cancer deaths. In males, chemists had the highest SMR, whereas in females, homemakers had the highest SMR. In males, the most common lung cancer cell type was squamous cell carcinoma, whereas in females adenocarcinoma was the most predominant type. The factors affecting the distribution of histologic lung cancer cell types were also investigated and discussed.

Adult↗

Survival of cells with bleomycin-induced chromosomal lesions in the cultured lymphocytes of lung cancer patients.

In a previous study of lung cancer, we showed that bleomycin, a radiomimetic agent, induced breaks preferentially on chromosomes 4 and 5. The molecular cytogenetic study reported here, using chromosome painting and G banding, was designed to assess whether the chromatid breaks induced by bleomycin could survive as chromosome-type aberrations after treated lymphocyte populations were allowed to recover in a drug-free medium for one or two cell generations and whether the survival rates of lesions on chromosomes 4 and 5 differed in cases with lung cancer and controls. The findings from 16 cases and 14 controls showed that in samples allowed to recover for 48 h, most aberrations were of the chromosome type. The proportion of chromosome 5 abnormalities surviving as chromosome-type aberrations was significantly higher in the cells of lung cancer cases (13.4%) than in controls (4.6%; P < 0.0001). However, no significant differences in survival of chromosome 4 abnormalities were detected between cases and controls. The proportions of chromosome 5q13-q22 abnormalities were 5.3% in the cases and 0.6% in the controls (P < 0.0001). 5q13-q22 regions encompassed 38.4% of all abnormalities on chromosome 5 in the cases but only 14.5% in the controls. Therefore, the survival rate of chromosome 5 lesions (especially those at 5q13-q22) in lymphocytes might be used as a biomarker to identify populations at high risk for lung cancer.

Antibiotics, Antineoplastic↗

Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice.

The receptor tyrosine kinase Flk-1 (ref. 1) is believed to play a pivotal role in endothelial development. Expression of the Flk-1 receptor is restricted to endothelial cells and their embryonic precursors, and is complementary to that of its ligand, vascular endothelial growth factor (VEGF), which is an endothelial-specific mitogen. Highest levels of flk-1 expression are observed during embryonic vasculogenesis and angiogenesis, and during pathological processes associated with neovascularization, such as tumour angiogenesis. Because flk-1 expression can be detected in presumptive mesodermal yolk-sac blood-island progenitors as early as 7.0 days postcoitum, Flk-1 may mark the putative common embryonic endothelial and haematopoietic precursor, the haemangioblast, and thus may also be involved in early haematopoiesis. Here we report the generation of mice deficient in Flk-1 by disruption of the gene using homologous recombination in embryonic stem (ES) cells. Embryos homozygous for this mutation die in utero between 8.5 and 9.5 days post-coitum, as a result of an early defect in the development of haematopoietic and endothelial cells. Yolk-sac blood islands were absent at 7.5 days, organized blood vessels could not be observed in the embryo or yolk sac at any stage, and haematopoietic progenitors were severely reduced. These results indicate that Flk-1 is essential for yolk-sac blood-island formation and vasculogenesis in the mouse embryo.

Animals↗

Structural characterization of a homophilic binding site in the neural cell adhesion molecule.

We have previously used synthetic peptides to identify a homophilic binding site between Lys-243 and Glu-252 (KYSFNYDGSE) in the third immunoglobulin-like domain of the chick neural cell adhesion molecule (NCAM). In this report, we show that the deletion of this decapeptide sequence from chick NCAM or the scrambling of the first 5 amino acid residues led to the abolition of the homophilic binding activity of NCAM, thus confirming the role of this sequence in NCAM-NCAM binding. To investigate the involvement of individual residues of this decapeptide in NCAM binding, competition experiments were carried out using peptide analogues with various amino acid substitutions. Substitution of both Lys-243 and Asp-249 with Ala or of the 3 aromatic residues with Ala led to a total loss of activity, highlighting the importance of these residues in NCAM binding. Site-directed mutagenesis was then employed to substitute individual amino acids within the decapeptide sequence with Ala. The homophilic binding activity of mutant NCAMs transiently expressed in COS-1 cells was determined using the NCAM-Covasphere binding assay. Substitution of the charged residues with alanine decreased NCAM binding activity, implicating electrostatic interactions in NCAM binding activity. Substitution of the aromatic residues Tyr-244 and Phe-246 with Ala abolished NCAM binding activity, suggesting that hydrophobic and/or aromatic interactions may play an important role in NCAM homophilic binding. Substitution of amino acids in the predicted beta-strand portion of the decapeptide with Pro, which would tend to disrupt beta-strand conformation, led to a substantial loss of activity. Thus, NCAM-NCAM binding may also depend on the beta-backbone structure of this site. These results are consistent with the involvement of multiple amino acids within the decapeptide sequence in NCAM homophilic interaction.

Amino Acid Sequence↗

Identification of a peptide sequence involved in homophilic binding in the neural cell adhesion molecule NCAM.

The neural cell adhesion molecule NCAM is capable of mediating cell-cell adhesion via homophilic interactions. In this study, three strategies have been combined to identify regions of NCAM that participate directly in NCAM-NCAM binding: analysis of domain deletion mutations, mapping of epitopes of monoclonal antibodies, and use of synthetic peptides to inhibit NCAM activity. Studies on L cells transfected with NCAM mutant cDNAs using cell aggregation and NCAM-covasphere binding assays indicate that the third immunoglobulin-like domain is involved in homophilic binding. The epitopes of four monoclonal antibodies that have been previously shown to affect cell-cell adhesion mediated by NCAM were also mapped to domain 3. Overlapping hexapeptides were synthesized on plastic pins and assayed for binding with these monoclonal antibodies. One of them (PP) reacted specifically with the sequence KYSFNY. Synthetic oligopeptides containing the PP epitope were potent and specific inhibitors of NCAM binding activity. A substratum containing immobilized peptide conjugates also exhibited adhesiveness for neural retinal cells. Cell attachment was specifically inhibited by peptides that contained the PP-epitope and by anti-NCAM univalent antibodies. The shortest active peptide has the sequence KYSFNYDGSE, suggesting that this site is directly involved in NCAM homophilic interaction.

Amino Acid Sequence↗

Properties of hemoglobin and dextran-hemoglobin rightshifted by oxidized inositol tetrakisphosphate.

Phytate was digested by wheat bran phytase to yield inositol tetrakisphosphate. Periodate-oxidized inositol tetrakisphosphate (oxyIP4) was coupled by means of reductive alkylation to hemoglobin and the covalent dextran-hemoglobin conjugate to yield the rightshifted (rs) compounds rsHb and rsDxHb, respectively. The variations of the oxygen dissociation curves of these molecules with pH and temperature were compared to those of hemoglobin. The variations with pH were found to be less pronounced for these rightshifted forms. An extensive decrease in the half-saturation oxygen tension was observed, however, with both rsHb and rsDxHb, as in the case of unmodified Hb. Modification of hemoglobin by oxyIP4 at the polyphosphate site was suggested by the lack of a further rightshifting effect of phytate on rsHb, and by the similarity between the difference spectrum of rs-methemoglobin and the difference spectrum induced by the addition of phytate.

Dextrans↗

The effects of zinc on the alveolar macrophage cytotoxicity due to Ni-coal dust.

The possibility of the combined effects on the alveolar macrophage cytotoxicity due to zinc and Ni-coal dust was studied. The parameters in the experiments were cellular ATP level in alveolar macrophage (AM), cellular K+ and Zn++ in AM and the surface morphological changes of AM. The experimental results showed that low zinc can protect the toxicity of Ni-coal dust on AM, and that the protective effects on the toxicity of Ni-coal dust by zinc are dependent on its dosage. The ATP level and K+ content in AM are sensitive indices in reflecting the cytotoxicity and can be used in detecting environmental toxic substances.

Adenosine Triphosphate↗