Production of native and modified recombinant Der p 1 molecules in tobacco plants.

BACKGROUND: As a complex molecule requiring post-translational processing, it has been difficult to produce the Der p 1 major allergen from the Dermatophagoides pteronyssinus house dust mite in a recombinant form. OBJECTIVE: Here, we tested whether transgenic tobacco plants are suitable to express Der p 1, either as a wild-type molecule or as variants lacking N-glycosylation sites (Gly(-)) and/or cysteine protease activity (Enz(-)). Methods Using Agrobacterium tumefaciens-based transformation, pro Der p 1 molecules bearing mutations within either the N-glycosylation sites (N34Q, N150Q) and/or the cysteine protease-active site (C132V) were expressed in tobacco plants. After purification by ion exchange chromatography, allergens were characterized using immunoblotting, circular dichroism (CD), as well as basophil and T lymphocyte stimulation assays. RESULTS: Four forms of recombinant Der p 1 (i.e. wild-type Gly(+)/Enz(+), as well as Gly(-)/Enz(+), Gly(+)/Enz(-) or Gly(-)/Enz(-) variants) were successfully expressed in tobacco leaves as pro Der p 1 molecules. Spontaneous cleavage of the pro-peptide was observed in tobacco leaf extracts for all forms of recombinant Der p 1 (r Der p 1). CD confirmed that all r Der p 1 molecules, with the exception of the Gly(-)/Enz(-) variant, exhibited secondary structures comparable to the natural protein. A cysteine protease activity was associated only with the Gly(+)/Enz(+) form. All these molecules exhibit a profile similar to natural Der p 1 with respect to IgE immunoreactivity, basophil activation and T cell recognition. CONCLUSION: A tobacco plant expression system allows the production of various forms of mature Der p 1, which could be used for diagnostic or immunotherapeutic purposes.

Protein analysis by mass spectrometry and sequence database searching: a proteomic approach to identify human lymphoblastoid cell line proteins.

Lymphoblastoid cell lines correspond to in vitro EBV-immortalized lymphocyte B-cells. These cells display a suitable model for experiments dealing with changes in protein expression occurring upon B-cell differentiation, after drug treatment, or after inhibition of some transcription factors. For all these reasons we have undertaken an effort aimed at developing a hematopoietic cell line protein two-dimensional electrophoresis (2-DE) database, containing B-lymphoblastoid 2-DE maps. In this work, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting analysis was adopted for protein identification. The peptide mass fingerprinting identification and the sequence coverage obtained on colloidal Coomassie blue (CBB) stained gel was close to that obtained using zinc-imidazole staining. Everything considered, CBB being more comfortable for subsequent spot manipulations, CBB staining was chosen for identification of a larger number of polypeptides. The results suggest that reticulation of the gel can interfere preventing the uptake of the enzyme during the in-gel digestion step. Consequently, low molecular mass proteins appear more difficult to identify by mass fingerprinting. Finally, the information provided in this study allows the construction of a new annoted reference map of human lymphoblastoid cell proteins. Among the identified proteins 60% were not yet positioned on 2-DE maps in three of the most important well-documented databases. The annoted map will be accessible via Internet on the LBPP server at URL:http:// www-smbh.univ-paris13.fr/lbtp/index.htm.

Absence of DNA adduct formation by phenobarbital, polychlorinated biphenyls, and chlordane in mouse liver using the 32P-postlabeling assay.

Phenobarbital (PB), polychlorinated biphenyls (PCBs), and chlordane (CLD) increase liver tumor incidences in rodents, and all are tumor promoters. Most indirect tests for DNA reactivity, including mutagenicity and chromosomal damage, have been negative with these agents. Consequently, the modes of action for tumorigenesis by these compounds are not believed to involve direct DNA reactivity; however, only limited information from direct tests is available for the lack of DNA adduct formation. PB, PCBs, and CLD were tested for DNA adduct formation in the liver of male and female B6C3F1 mice after either single or 2-week dietary exposures. Single gavage dose levels were as follows: PB, 200 mg/kg; PCBs, 50 mg/kg; and CLD, 50 mg/kg. Dietary dose levels were as follows: PB, 1000 ppm; PCBs, 200 ppm and CLD, 200 ppm. Animals were killed 24 h following the end of test-substance administration. DNA was extracted from the liver, and DNA adduct concentrations were enriched using either 1-butanol extraction of adducted nucleotides or nuclease P1 digestion of unadducted nucleotides. Using this protocol, none of the three test compounds produced DNA adducts detected by 32P-postlabeling. Similar negative results were obtained for DNA from the livers of both male and female mice receiving either single or 2-week exposures. The two positive controls, benzidine for the 1-butanol extraction procedure and 2-acetylaminofluorene for the nuclease P1 procedure, showed the expected patterns of DNA adducts. These results support the conclusion that the carcinogenicity of PB, PCBs, and CLD in experimental animals is not the result of direct DNA reactivity, but involves epigenetic mechanisms.

Comparison of DNA reactivity of the polyphenylethylene hormonal agents diethylstilbestrol, tamoxifen and toremifene in rat and hamster liver.

The polyphenylethylene estrogenic drug diethylstilbestrol and a structural analogue tamoxifen have been found to be hepatocarcinogenic in female rats, whereas another analogue, toremifene, did not induce liver tumors. The 32P post-labelling technique for detection of DNA adducts was used to investigate the DNA reactivity of these three hormonal agents in the livers of female Sprague-Dawley rats and Syrian hamsters. Adducts were quantified using a radioanalytic imaging system in comparison with the standard Cerenkov assay. With administration of the chemicals at several doses by daily gavage to rats for 10 days and to hamsters for 7 days, tamoxifen was found to produce five adducts in rat liver and six adducts in hamster liver. The amounts of adducts were dose related from 10 to 90 mumol/kg per day in rats and from 17 to 160 mumol/kg per day in hamsters. The two methods of quantification yielded comparable results. Under these conditions, neither toremifene nor diethylstilbestrol produced adducts in rats and diethylstilbestrol produced none in hamsters. We conclude that tamoxifen is highly DNA reactive in the species studied and that this is likely to be involved in its strong carcinogenicity in rat liver.

Concomitant cellular expression of heat shock regulated genes of hepatitis B virus surface antigen and of human growth hormone by a NIH-3T3 cell line.

A plasmid carrying a DNA fragment of hepatitis B virus, coding for the pre-S2 and the entire S region of the surface antigen (HBsAg), placed under the control of the promoter of the human 70 kDa heat shock protein gene (hsp70), was introduced into Line 6, a recombinant cell line that was selected from NIH-3T3 cells previously transfected with a similar construct coding for the human growth hormone cDNA gene (chGH) and with the plasmid pEJ carrying the Ha-rasEJ activated cellular oncogene. The resulting cell line, EMS8, expressed: (1) hsp70/HBsAg and hsp70/hGH hybrid genes, (2) the human Ha-rasEJ oncogene, and (3) the neomycin resistance gene, the two last plasmid markers being used for cell selection. EMS8 cells were able to carry out post-translational modifications of the middle M and the major S envelope proteins of HBV, such as assembly and glycosylation. Accordingly, the cells synthesized and secreted both free and glycosylated M and S viral proteins, and the human growth hormone protein. In addition concomitant expression of HBsAg and hGH proteins as well as their mRNA were detected in EMS8 cells at least up to 72 hr after heat induction instead of 24 hr in the case of hGH in Line 6 cells.

Expression of heat shock-regulated human growth hormone genes containing or lacking introns by NIH-3T3 and Wish cell lines.

A plasmid containing the complete genomic DNA of the human growth hormone (ghGH) comprising four introns and driven by the human promoter of the human gene of the 70 kDa heat shock protein (hsp70) has been used to transfect mouse NIH-3T3 and human Wish cells. Selected cell lines were characterized for stable hGH secretion. Similarly in the same NIH-3T3 cells, the stable expression of the same plasmid construct, but containing the complementary DNA of the hGH gene (chGH), was compared in terms of the effect of introns on heterologous protein synthesis. Genomic hGH recombined cells synthetized, in a heat regulated fashion, matured hsp70/hGH hybrid mRNA able to drive the secretion of a 22 kDa polypeptide. Like the natural hGH, this polypeptide expressed the functional hormonal activity of prolactin on casein secretion by mammary cells. The time course of hGH secretion was prolonged in ghGH transcripts, while that of mRNA degradation appeared delayed, especially in Wish cells, as compared to chGH expression. In the human Wish cells the decay of endogenous hsp mRNA has been compared to that of recombinant hsp mRNA, demonstrating that this human hsp70/hGH hybrid mRNA was present in the cytoplasm during a longer period than the human endogenous hsp70 mRNA. In conclusion, similar levels of expression and resulting gene products were expressed from the chGH or the ghGH gene in an inducible manner.

[Immortalization of cultured cells by the Ha-rasEJ oncogene and expression of a gene of biogenetic value]. / Immortalisation de cellules en culture par l'oncogène Ha-rasEJ et expression d'un gène d'intérêt biogénétique.

The double recombination into NIH-3T3 cells of the cloned Ha-rasEJ oncogene and the cDNA gene of the human growth hormone (hGH) under the control of a heat inducible promoter (hsp70) allowed hGH production either in vitro using the mass culture of engineered cells in biogenerators, or in vivo after xeno-transplantation of the cells into an animal host. Therefore the in vivo synthesized hGH induced the production of anti-hGH polyclonal antibodies.

Maintenance of liver function in long term culture of hepatocytes following in vitro or in vivo Ha-rasEJ transfection.

Collagenase isolated rat hepatocytes were transfected with liposome encapsulated pEJ (LE-pEJ), a plasmid carrying the human cellular activated Ha-rasEJ oncogene. A proliferative cell line was cloned from these cells transfected in vitro. It secreted per day 0.87 micrograms albumin and 0.32 microgram transferrin per 10(6) cells, and 11.06 nmol free and conjugated bile acids (BA) per mg protein. Also, it metabolized 2-acetylaminofluorene (2-AAF) into N- and ring-hydroxylated metabolites and 2-aminofluorene at rates of 1.50, 9.73, and 1.98 nmol/mg cell protein/24 hr, respectively. Rats were i.v. injected with both LE-pEJ and LE-p17hGHneo carrying the hGH cDNA gene, and secreted hGH in the plasma which induced the synthesis of anti-hGH antibodies. A cell line was cloned from cultures of primary hepatocytes isolated from the liver of transfected rats. After 2 to 3 months in culture, this cell line secreted per day 18.9 micrograms albumin and 11.0 micrograms transferrin per 10(6) cells, 38.75 nmol total BA per mg cell protein, and up to 31 ng hGH per 10(6) cells without cloning hGH recombinant cells. A 24 hr control culture of primary hepatocytes isolated from non transfected rats secreted 25.5 micrograms albumin and 11.7 micrograms transferrin per 10(6) cells, and produced 21.64 nmol total BA and 2.13 nmol N-OH-2-AAF per mg cell protein. Hence, Ha-rasEJ transfection of either hepatocytes in vitro or liver cells in vivo, initiated cell cycles leading to presumptive proliferating hepatocytes which express liver function.

Methylation state and DNase I sensitivity of chromatin containing Moloney murine leukemia virus DNA in exogenously infected mouse cells.

The nature of Moloney murine leukemia virus (M-MuLV)-specific proviral DNA in exogenously infected mouse cells was studied. M-MuLV clone A9 cells, NIH-3T3 fibroblasts productively infected with M-MuLV, were used. These cells contain 10 to 15 copies of M-MuLV proviral DNA. The state of methylation of M-MuLV proviral DNA was examined by cleaving A9 cell DNA with restriction endonucleases which have the dinucleotide CpG in their cleavage sequences. Analysis with such enzymes, which recognized nine different sites in M-MuLV DNA, indicated that most if not all of the M-MuLV proviruses in A9 cells were completely unmethylated. An individual proviral integration was examined, using as probe adjacent single-copy cellular sequences. These sequences were obtained from a lambda phage recombinant clone containing an M-MuLV provirus from the A9 cells. This individual integration also showed no detectable methylation. In contrast, endogenous MuLV-related sequences present in NIH-3T3 cells before infection were largely methylated. The configuration chromatin containing M-MuLV proviruses was also investigated by digesting A9 nuclei with DNase I, followed by restriction analysis of the remaining DNA. Endogenous MuLV-related DNA was in chromatin relatively resistant to DNase I digestion, whereas the majority of M-MuLV-specific proviruses were in domains of intermediate DNase I sensitivity. Two proviral copies hypersensitive to DNase I digestion were identified. Analogy to the DNase I sensitivity of expressed and nonexpressed globin genes suggested that the proviral copies containing DNase I-hypersensitive sites were transcribed.