Use of adenovirus vectors for functional gene analysis in the chicken chorioallantoic membrane

The chicken chorioallantoic membrane (CAM) assay represents one of the most widely used in vivo screening assay for genes with angiogenic (blood vessel-inducing) or angiostatic (inhibition of vessel formation or their destruction) activities. Here we show that adenovirus gene transfer vectors infect cells in the CAM and lead to expression of the viral transgene. Furthermore, infection with an adenovirus vector containing the human vascular endothelial growth factor gene induced the formation of new blood vessels. This improved method saves a considerable amount of time in the identification of genes that can influence blood vessel formation because the expensive and time-consuming production and purification of recombinant protein can be omitted.     

Regional hypoxia elicits regional changes in chorioallantoic membrane vascular density in alligator but not chicken embryos

Hypoxic incubation increases vascularization in the chick chorioallantoic membrane (CAM). The effect of regional hypoxia on the vascular density of American alligator (Alligator mississippiensis) and chicken (Gallus gallus) CAMs was studied to determine if hypoxic proliferation of blood vessels is localized or global across the CAM. Eggs were incubated under normoxic conditions with a portion of the eggshell covered with non-toxic beeswax to induce external regional hypoxia. CAMs were examined under a microscope with a 'bulls eye' coverslip and a vascular density index (VDI) was determined. The hypoxic portions of the alligator CAMs were more vascular than the normoxic portions (VDI = 200.9 versus 157. 8, respectively). Presumably this response is maladaptive by causing increased blood flow to the poorly oxygenated portions, i.e. increased 'shunt'. Thus, we hypothesize increased vascularity due to hypoxic incubation is due to local release and subsequent rapid local breakdown or uptake of angiogenic factors. In contrast, the hypoxic and normoxic portions of the chick CAMs exhibited virtually no difference in VDI (VDI= 211.5 versus 217.9, respectively). We suggest the air cell and air space of the chicken eggs allows for circulation of gas in ovo, eliminating the possibility of regional internal hypoxia.     

Construction of a recombinant eukaryotic expression plasmid containing human PDLIM2 gene and its biological activity

In order to obtain a full-length expression plasmid for human PDLIM2 gene, fragment amplification was used to clone its full-length coding sequence (CDS) region. The amplified PCR product was then digested and inserted into the pMD 18-T vector and subcloned into the pIRES2-EGFP plasmid to form the pIRES2-EGFP-PDLIM2 eukaryotic expression vector. After it was transfected to the bladder cancer cell line, BIU-87, the biological activities of high expression were verified by RT-PCR and Western blotting. Meanwhile the mRNA and protein expressions of p65 were detected. Finally we analyzed the effect of overexpressed PDLIM2 on BIU-87 cell proliferation. In conclusion, a recombinant eukaryotic expression vector pIRES2-EGFP-PDLIM2 containing the complete CDS region of PDLIM2 was successfully constructed. PDLIM2 negatively regulated p65 expression and inhibited BIU-87 cell proliferation. We laid the foundations for further research into the function of the PDLIM2 gene in bladder cancer.     

Nitration of cytoskeletal proteins in the chicken embryo chorioallantoic membrane

Protein tyrosine nitration is one of the post-translational modifications that alter the biological function of proteins. Two important mechanisms are involved: peroxynitrite formation and myeloperoxidase or eosinophil peroxidase (EPO) activity. In the present work we studied the nitration of proteins in the in vivo system of chicken embryo chorioallantoic membrane (CAM). 3-Nitrotyrosine was detected only in the insoluble fraction of the CAM homogenate. By immunoprecipitation, Western blot analysis, and double immunofluorescence, we identified two major polypeptides that were nitrated: actin and alpha-tubulin. Quantification of actin and alpha-tubulin nitration revealed that they are differentially nitrated during normal development of the chicken embryo CAM. After irradiation, although they were both increased, they required different time periods to return to the physiological levels of nitration. It seems that both peroxynitrite formation and EPO activity are involved in the in vivo tyrosine nitration of cytoskeletal proteins. These data suggest that tyrosine nitration of cytoskeletal proteins has a physiological role in vivo, which depends on the protein involved and is differentially regulated.     

Construction and properties of a recombinant plasmid containing gene 32 of bacteriophage T4D

This paper describes the construction and characterization of a chimeric plasmid that encodes the single-stranded DNA-binding protein of bacteriophage T4D (the product of gene 32). The plasmid contains a 2·6 × 10³ base HindIII segment of T4 DNA that includes genes 59 and 32 as well as a portion of gene 33. Isolation of bacteria carrying the recombinant plasmid became possible when the segment of phage DNA contained an amber mutation in gene 32. This suggests that a functional gene 32 is deleterious to the cell. Using antibody to gene 32 protein, we have been able to demonstrate expression of the plasmid-borne gene 32 in uninfected bacteria. Deletion variants of the gene 32 plasmid have been constructed in vitro. These have been used to align the genetic map of the region with the restriction map and to study phage gene expression from the plasmid in both infected and uninfected cells. In phage-infected cells the level of functional gene 32 product regulates the efficiency of translation of its own messenger RNA. We also observe such self-regulation for gene 32 present on the plasmid.     

Suppression of growth and cancer-induced angiogenesis of aggressive human breast cancer cells (MDA-MB-231) on the chorioallantoic membrane of developing chicken embryos by E-peptide of pro-IGF-I

E-peptide of the pro-Insulin-like growth factor-I (pro-IGF-I) is produced from pre-pro-IGF-I by proteolytic cleavage in the post-translational processing. Previous in vitro studies conducted in our laboratory showed that Ea4-peptide of rainbow trout (rt) pro-IGF-I or Eb-peptide of human (h) pro-IGF-I exhibited activities including induction of morphological differentiation, inhibition of anchorage-independent cell growth and suppression of invasion of several well established human cancer cell lines such as MDA-MB-231, HT-29, SK-N-F1, and HepG-2 (Chen et al. [2002] Gen Comp Endocrinol 126:342-351; Kuo and Chen [2002] Exp Cell Res 280:75-89). Seeding of aggressive human breast cancer cells, MDA-MB-231, on the chorioallantoic membrane (CAM) of 5 days old chicken embryos resulted in rapid growth and invasion of the cells and induction of blood vessel formation around the MDA-MB-231 cell mass in the chicken embryos. The invasion of MDA-MB-231 cells in the chicken embryos was further confirmed by immunocytochemistry. The rapid growth and invasion of MDA-MB-231 cells and the induction of blood vessel formation by MDA-MB-231 cells on chicken CAM are inhibited by treatment with a single or multiple doses of rtEa4- or hEb-peptide. Furthermore, a dose-dependent inhibition of angiogenesis by rtEa4- or hEb-peptide was also demonstrated by the chicken CAM assay. Results of microarray analysis of human gene chips (containing 9,500 unique cDNA clones) and confirmation by comparative real-time RT-PCR analysis showed that a group of genes related to cancer cell activities are up- or down-regulated in MDA-MB-231 cells transfected with a rtEa4-peptide gene. Together these results confirm the anti-tumor activity of rtEa4- and hEb-peptides, and further suggest that these peptides could be developed as therapeutics for treating human cancers.     

Phenotypic reversal in dam mutants of Escherichia coli K-12 by a recombinant plasmid containing the dam+ gene.

A recombinant plasmid, pMQ3, carrying the dam gene of Escherichia coli K-12, was constructed and transformed into dam+ and dam- strains. Both dam- and dam+ strains containing pMQ3 showed a wild phenotype for all traits, including mutation rate, except for a 10-fold increase in DNA adenine methylase activity.     

转染hTERT基因的大鼠神经干细胞生物学特性的研究

目的 本试验旨在研究hTERT基因所表达的蛋白对NSCs生物学特性的影响。方法 本试验通过脂质体转染法将构建的重组质粒pEGFP-N1-hTERT转染到大鼠胎儿NSCs中,对转染的NSCs进行体外诱导分化试验、裸鼠致瘤性试验、RT-PCR、Western-blot和流式细胞仪分析。结果 转染的NSCs在体外培养中仍具有干细胞的生长特性和多潜能性、无致瘤性,转染重组质粒的NSCs中,存在hTERT基因mRNA的转录产物和融合蛋白hTERT-GFP的表达。结论 hTERT基因对神经干细胞端粒酶活性有上调作用,可促进NSCs的体外增殖和永生化趋势。     

Isolation and characterization of recombinant clones containing the chicken adult beta-globin gene.

We have isolated and characterized two independent clones containing the chicken adult beta-globin gene. Each clone contains a 6.2-kilobase-pair Eco RI restriction fragment of chicken erythrocyte DNA inserted into the vector, lambda gtWES . lambda B. The orientation of the inserted fragment is opposite in the two clones. Characterization of the clones by electron microscopic R-loop studies, by restriction enzyme mapping, and by filter hybridization shows that the adult beta-globin gene is interrupted by at least one small and one large intervening sequence. In addition to the complete adult beta-globin gene, at least part of a second beta-globin-like gene was identified about 2.7 kilobase pairs from the 3'-end of the adult gene. The two independent clones, while very similar, do differ at two Msp I restriction endonuclease sites in regions flanking the adult beta-globin gene.     

Fate in soil of a recombinant plasmid carrying aDrosophila gene

A recombinant plasmid (C357; 3.5 Mdal) containing heterologous DNA (pBR322 [2.6 Mdal] with cDNA for an egg yolk protein fromDrosophila grimshawi) inEscherichia coli strain HB101 survived in and was recovered on selective media from sterile and nonsterile soil during 27 days at frequencies similar to those of theE. coli(pBR322) system. In sterile saline, the numbers of all cells decreased during 34 days, but the numbers of the plasmidless host declined less. There was no selective loss of the heterologous DNA in either soil or saline, as determined by colony hybridization with a³²P-labeled DNA probe for the cDNA, but the HB101(C357) appeared to be less able than HB101(pBR322) to cope with conditions of starvation. These results suggested that nonessential eucaryotic DNA inserted into plasmid DNA has little effect on the survival in soil or saline of the bacterial host and the maintenance of the vector.     

Induction of angiogenesis by mixtures of two angiogenic proteins, angiogenin and acidic fibroblast growth factor, in the chick chorioallantoic membrane

The chick chorioallantoic membrane assay was employed to assess the angiogenic response induced by mixtures of human angiogenin with bovine heparin-binding acidic fibroblast growth factor. Statistical evaluation of data accumulated at several molar ratios of the two proteins indicate that the angiogenic activity observed is neither an additive nor a synergistic resultant of the activities of the proteins separately. The possibility exists, however, that at an approximately 1:1 mole ratio an apparent inhibitory effect can be observed. Mechanisms which could underlie such observed effects are discussed.     

The use of cell cultures from the chorioallantoic membrane of chick embryos for studies of chlamydiae

Cells of the chorioallantoic membrane of chick embryos were processed into monolayer cell cultures by a recently described technique of Cursiefen and Brecht. The cultures provided a sensitive substrate for the quantification of infectivity of C. psittaci strain 6BC and meningo-pneumonitis.     

Characterization of the protein expressed in Escherichia coli by a recombinant plasmid containing the Bacillus megaterium cytochrome P-450BM-3 gene

Summary In two previous reports (Narhi LO, Fulco AJ, J. Biol. Chem. 261: 7160–7169, 1986; Ibid., 262: 6683–6690, 1987) we described the characterization of a catalytically self-sufficient 119000-dalton P-450 cytochrome that was induced by barbiturates in Bacillus megaterium. In the presence of NADPH and O₂, this polypeptide (cytochrome P-450_BM-3) catalyzed the hydroxylation of long-chain fatty acids without the aid of any other protein. The gene encoding this unique monooxygenase was cloned into Escherichia coli and the clone harboring the recombinant plasmid produced a protein that behaved electrophoretically and immunochemically like the B. megaterium enzyme (Wen LP, Fulco AJ, J. Biol. Chem. 262: 6676–6682, 1987). We have now compared authentic P-450_BM-3 from B. megaterium and putative P-450_BM-3 isolated from transformed E. coli and have found them to be indistinguishable with respect to chromatographic and electrophoretic behavior, reaction with specific antibody, prosthetic group (heme, FAD and FMN) analyses, spectra, enzymology, limited trypsin proteolysis and partial amino acid sequencing. We thus conclude that the P-450 cytochrome expressed by the transformed E. coli is essentially identical to native P-450_BM-3 induced by barbiturates in B. megaterium. The evidence furthermore suggests that the primary amino acid sequence of this complex protein is alone sufficient to direct the proper integration of the three prosthetic groups and to specify folding of the polypeptide into the correct tertiary structure.Abbreviations SDS Sodium Dodecylsulfate - PAGE Polyacrylamide Gel Electrophoresis - HPLC High Performance Liquid Chromatography     

Calcium transport and related functions in the chorioallantoic membrane of cultured shell-less chick embryos

In order to investigate the influence of the egg shell on the process of shell calcium mobilization by the chorioallantoic membrane (CAM), chick embryos were maintained in long-term cultures in vitro without the shells. The shell-less embryos were severely calcium deficient and showed signs of retarded development and anomalous skeletal calcification. Throughout development, calcium transport and calcium-binding protein (CaBP) activities were diminished in the CAM of shell-less embryos as compared to those of control embryos which developed in ovo. The levels of developmentally expressed carbonic anhydrase activity remained, however, similar. By means of a single radial immunodiffusion assay of CaBP using a specific anti-CaBP antiserum, the level of immunoreactive CaBP was found to be significantly increased in the CAM of the shell-less embryos. These studies indicate that the CAM of chick embryos cultured under shell-less conditions is defective in calcium transport, probably as a result of the expression of an inactive form of the CaBP.     

Early embryogenesis study on a dimethoate containing formulation and Cd-sulphate in chicken embryos

The toxic effects of a widely used organophosphate insecticide (BI 58 EC containing 38% dimethoate as active ingredient) applied alone or in combination with cadmium sulphate modelling the heavy metal load of the environment were studied on chicken embryos in the early phase of development. Solutions and emulsions of different concentrations were made from the test materials and injected in 0.1 ml volume into the air space of eggs on the first day (day 0) of incubation. Subsequently, on days 2 and 3 of incubation permanent preparations were made from the embryos in order to study the early developmental stage. Embryos fixed on slides and stained with osmium tetroxide solution were studied under light microscope. Summarising the findings, it can be established that the embryotoxicity increased after the simultaneous administration of Cd-sulphate and 38% dimethoate containing insecticide formulation compared to the control or the individually treated groups.     

Stability of a recombinant plasmid carrying a-amylase gene in Bacillus subtilis

Plasmid pSB6 is a streptococcal recombinant plasmid carrying the a-amylase gene of Bacillus amyloliquefaciens and the chloramphenicol resistance gene. The segregational and structural instabilities of this plasmid were examined under non-selective conditions in Bacillus subtilis. These instabilities were modelled according to a kinetic expression derived from the difference in the growth between plasmid-bearing and plasmid-free cells. This plasmid showed slight segregational instability and much higher levels of structural instability under the conditions examined.     

Stability of a recombinant plasmid carrying a-amylase gene in Bacillus subtilis

Plasmid pSB6 is a streptococcal recombinant plasmid carrying the a-amylase gene of Bacillus amyloliquefaciens and the chloramphenicol resistance gene. The segregational and structural instabilities of this plasmid were examined under non-selective conditions in Bacillus subtilis. These instabilities were modelled according to a kinetic expression derived from the difference in the growth between plasmid-bearing and plasmid-free cells. This plasmid showed slight segregational instability and much higher levels of structural instability under the conditions examined.     

Expression, purification and characterization of recombinant human angiogenin in Pichia pastoris

The potential of angiogenin (Ang) for clinical use has been highlighted in view of its important roles in inducing angiogenesis, facilitating cell proliferation, and inhibiting cell apoptosis. To produce soluble, correctly folded recombinant protein with a high yield, a DNA fragment encoding human Ang was inserted into eukaryotic expression vector pPIC9 and transformed into Pichia pastoris. The expression of recombinant human Ang (rhAng) accounted for about 70% of total secreted proteins. Purifying the Ang from the culture supernatant yielded 30 mg/L at 90% purity by chromatography with a SP Sepharose FF column. Biological assays indicated that rhAng can induce new blood-vessel formation, promote HeLa cell proliferation, increase Erk1/2 phosphorylation, and upregulate c-myc expression. Preparation of bioactive rhAng might lay the basis for further functional study, and might provide an effective strategy for large-scale production of soluble human Ang.