Biomedical subjects

M Charlier

Publications and source records attributed to M Charlier.

At least 55 records · Page 3Linked to original sources

PubMed1993

Cloning and structural analysis of four genes encoding interferon-omega in rabbit.

By using an ovine interferon-tau (IFN-tau) cDNA probe, four recombinant phages were isolated from a rabbit genomic library and sequenced from nucleotides -450 to 1,300 relative to the CAP site. Each of the four rabbit genes contains an open reading frame of 595 nucleotides and code for proteins that exhibit structural characteristics of the interferon-omega (IFN-omega) family. They display more than 98% identity in their coding regions. The deduced amino acid sequences share > 96% sequence similarity. In contrast, the 5' and 3' noncoding regions have diverged considerably (approximately 50% identity). Amino acid comparisons of rabbit IFN-omega with IFN-omega of other species reveal the highest degree of identity with human (72%), followed by porcine (68%) IFN-omega. Rabbit IFN-omega displays only 57% sequence similarity with ovine IFN-tau. The coding regions of the four genes subcloned in a cytomegalovirus eukaryotic expression vector and transfected in monkey COS-7 cells direct the production of proteins that protect bovine and rabbit cells against vesicular stomatitis virus infection, thus demonstrating that these genes encode fully active IFN proteins. The expression of these genes was studied in Sendai-induced rabbit leukocytes. A single band of poly(A)+RNA hybridized with a rabbit IFN-omega probe under stringent conditions, whereas no IFN-omega transcript was detected with RNA isolated from uninduced leukocytes. Southern blot analysis suggest the existence of at least eight IFN-omega genes or pseudogenes in the rabbit genome.

Amino Acid Sequence↗

PubMed1992

Addition of a dipeptide spacer significantly improves secretion of ovine trophoblast interferon in yeast.

Yeast has been analysed for its potential to secrete an ovine member of the type-I interferon (IFN) family, trophoblastin (oTP-1). The processing potential of the yeast KEX2 gene product (KEX2p) was evaluated using gene oTP-1 fused to the pre-pro sequence encoding the pre-pro peptide of the yeast alpha-factor precursor. High-level accumulation of nonprocessed (unmatured) recombinant oTP-1 (re-oTP-1) was observed in the medium. In order to short-circuit the limiting activity of KEX2p and to obtain a fully matured re-oTP-1, secretion was directed using a pre::oTP-1 fusion, relying only on signal peptidase-dependent processing. However, secretion of oTP-1 was impaired. High-level secretion was restored when the gene product contained a peptide spacer between oTP-1 and the signal peptidase cleavage site. The oTP-1 variant was shown to have an extended N terminus. An N-extended form was examined further and shown to have the correct size. Surprisingly, the variant retained its in vitro and in vivo biological activities. This system is likely to represent a general method for high-level secretion of type-I IFNs.

Amino Acid Sequence↗

PubMed1992

Metal ions protect DNA against strand breakage induced by fast neutrons.

Single and double strand breaks (SSB and DSB) are induced by fast neutrons in plasmid (pBR322) DNA in 1 mM potassium phosphate buffer (pH 7.25). Increasing the concentration of monovalent (Na+, Cs+, Li+), divalent (Mg2+, Ca2+) and trivalent (Al3+, Co3+ (NH3)6) metal cations strongly decreases the yield of DSB. The extent of the observed protection depends on the valence of the cation. The production of SSB is only slightly decreased, except for Al3+ and Co3+ (NH3)6, whose effects are particularly large (complete protection at 1 and 0.1 mM respectively). Circular dichroism spectra show that Al3+ induces an important structural change of DNA at the ion concentration where the protection becomes total. This change is probably a condensation (collapse), as in the well-known case of Co3+ (NH3)6. Our results suggest two mechanisms of protection by metal ions: (i) the induction of structural changes of DNA, that render less accessible the critical sites of attack by OH. radicals; and (ii) the stabilization of the double helical regions between two close-set nicks on opposite strands, that hinders the effective double strand breakage of DNA.

Cations↗

PubMed1991

Expression of a biologically active ovine trophoblastic interferon using a baculovirus expression system.

Ovine trophoblast protein (oTP) an embryonic interferon, which plays a key role in maternal recognition of pregnancy, has been expressed in insect cells using a baculovirus expression system. A cDNA coding for oTP was inserted downstream of the strong polyhedrin promoter. Cells infected with recombinant virus produced biologically active oTP and greater than 90% was secreted into the culture medium during infection. High amount of antiviral activity were produced (up to 5 x 10(5) IU per ml of culture medium). Recombinant oTP (roTP) was purified by immunoaffinity chromatography and found to be identical to authentic oTP with respect to molecular mass and N-terminal amino acid sequence.

Amino Acid Sequence↗

PubMed1991

Determination of the DNA-interacting region of the archaebacterial chromosomal protein MC1. Photocrosslinks with 5-bromouracil-substituted DNA.

Protein MC1 is the major chromosomal protein in methanosarcinaceae. Using photochemical crosslinking on 5-bromouracil-substituted DNA, we identified the region of the protein that interacts with it. This region is located in the C-terminal part of the polypeptide chain, and the crosslinked amino-acids are in the region 74-86. Tryptophan 74 is one of the amino-acids crosslinked to DNA.

Archaeal Proteins↗

PubMed1991

The bovine alpha-lactalbumin promoter directs expression of ovine trophoblast interferon in the mammary gland of transgenic mice.

A hybrid construct derived from ovine trophoblastin cDNA and bovine alpha-lactalbumin-encoding gene, was injected into the pronuclei of mouse eggs. In one of the resulting transgenic mouse lines, expression of the hybrid construct was detected and found to be limited to the mammary gland of lactating females which secreted active ovine trophoblastin. This strongly suggests that important cis-acting DNA sequences involved in tissue-specific expression of the bovine gene are located within the second half of the 3' untranslated region, or/and the proximal 5' and 3' regions flanking the transcriptional unit.

Animals↗

PubMed1991

Co-expression of the proto-oncogene fos (c-fos) and an embryonic interferon (ovine trophoblastin) by sheep conceptuses during implantation.

Expression of the c-fos proto-oncogene by ovine conceptuses was analyzed by Northern and slot blots and indirect immunohistofluorescence in relation to the expression of the embryonic interferon-alpha (oTP) during implantation. c-fos was expressed initially in the trophoblast, and then in the allantois, when this tissue began to develop (day 17). In the embryonic tissues, the c-fos proto-oncogene was weakly expressed up to day 22 and increased thereafter. In the trophoblast, the expression of c-fos proto-oncogene was transient, occurring when the oTP gene was transcribed at a maximal level at the beginning of implantation (days 14-15), and decreased thereafter, following the pattern of oTP gene expression. This decline is due essentially to the arrest of c-fos and oTP gene expression by the trophoblastic cells which established cellular contacts with the uterine epithelium during the implantation process.

Actins↗

PubMed1991

Cloning and structural analysis of two distinct families of ovine interferon-alpha genes encoding functional class II and trophoblast (oTP) alpha-interferons.

Ovine trophoblast protein (oTP) is a polypeptide secreted by ovine trophectoderm from day 11 to 21, which plays a key role in maternal recognition of pregnancy. Structural analyses established that oTP shares extensive homology with class II alpha-interferon (IFN-alpha II) subfamily. Previous screening of an ovine genomic DNA library probed with an oTP cDNA incidently resulted in the isolation of a functional IFN-alpha II gene and two relevant pseudogenes, as shown by sequence analysis and study of expression in eukaryotic COS cells. The expected oTP gene together with a cognate pseudogene was successfully isolated from the series of clones selected from another genomic library probed with the oTP cDNA, using two specific oligonucleotides, each one complementary to a region of oTP cDNA with little homology with the IFN-alpha II gene and related pseudogenes. Southern blotting of ovine genomic DNA indicated the existence of at least five trophoblast IFN-alpha genes or pseudogenes. Nucleotide sequence comparisons showed that the oTP gene exhibits a higher homology (90%) with bovine trophoblast IFN gene (Stewart et al. (1990) J. Mol. Endocrinol. 4, 275-282) than with oIFN-alpha II gene (70%), thus providing evidence that embryonic IFNs constitute a distinct subfamily of IFN-alpha s.

Amino Acid Sequence↗

PubMed1991

DNA radiolysis by fast neutrons. II. Oxygen, thiols and ionic strength effects.

Plasmid DNA was irradiated with fast neutrons, and the protection by cysteamine against strand breakage (ssb and dsb) was evaluated in the presence and absence of oxygen. In the absence of cysteamine no radiosensitizing effect of oxygen was observed. In anoxia the protection factors, PF(ssb) and PF(dsb) of 1 mM cysteamine (in 50 mM potassium phosphate solution) were lower than the PFs observed with gamma-irradiation. The results agree with the radical repair model, in which the thiol competes with the oxygen produced inside the anoxic neutron-irradiated system, according to the 'oxygen-in-the-track' hypothesis. At low ionic strength in air-saturated solutions, positively charged cysteamine protects more efficiently than negatively charged thiolactate. The dependence of the PFs on the charge of the thiol can be explained by the condensation of counter-ions and depletion of co-ions around DNA, predicted by Manning's theory. Based on the same theory, we propose here an explanation of the ionic strength or by thiolactate at low ionic strength is largely due to scavenging of OH. radicals in the bulk solution. At low ionic strength the PF(ssb) of cysteamine is higher for neutrons than for gamma-irradiation. This might suggest different primary lesions for the two types of irradiation.

Cysteamine↗

PubMed1991

Photochemical cross-linking of the cyclic adenosine 3',5' monophosphate receptor protein to Escherichia coli 5-bromouracil-substituted DNA. Role of the effectors.

Cyclic AMP receptor protein (CRP) is a regulatory protein implicated in the transcription of several operons in Escherichia coli. Its activity is modulated by effectors, such as cAMP or cGMP, which could induce (or not) structural changes in the protein, and activate (or not) the transcription. CRP can bind non-specifically to DNA, and we investigated the photocross-linking of the protein to E. coli 5-bromouracil-substituted DNA, in the absence and in the presence of effectors. The photochemistry of the protein alone, under the conditions used for the cross-linking reaction, was studied. We show that tryptophyl residues are more photoreactive than tyrosyl ones. Photocross-linking of the protein implicates only one of the two subunits, and the rate of the reaction is not modified upon cAMP binding. Binding of cGMP reduces the rate of photocross-linking by a factor of two. These new results show that the protein in the CRP-cGMP complex behaves differently from the free protein.

Bromouracil↗

PubMed1990

DNA radiolysis by fast neutrons.

The effects of fast neutron irradiation on DNA were studied using DNA of the pBR322 plasmid (4362 base pairs), and the results compared to those obtained with 60Co gamma rays. Irradiation of the plasmid DNA in solution with a neutrons beam (p34+Be) of the CERI (CNRS Orléans) cyclotron (with a flat energy spectrum from 34 MeV to low energies) results in half the yield of single-strand breaks (ssb), and 1.5 times higher yield of double-strand breaks (dsb) for neutrons as compared to gamma-rays. Possible specificity of the neutron-induced breaks was examined: the scavenging of OH. radicals by 0.1 mol dm-3 ethanol inhibits all neutron-induced ssb, but only 85 per cent of the dsb. For gamma-irradiation, both ssb and dsb are completely inhibited in these conditions. These results suggest at least three different origins for neutron-induced dsb. The occurrence of around 30 per cent of dsb can be explained by a radical transfer mechanism (proposed by Siddiqi and Bothe (1987) for gamma-irradiation). Around 55 per cent of dsb may be due to the non-random distribution of radicals in high-density tracks of the secondary particles of neutrons, which results in a simultaneous attack of the two strands by OH. radicals. These first two processes are both OH.-mediated and thus are sensitive to ethanol. The direct effect of fast neutrons and their secondaries (recoil protons, alpha-particles and recoil nuclei) can account for the remaining 15 per cent of dsb, not inhibited by 0.1 mol dm-3 ethanol.

Cobalt Radioisotopes↗

PubMed1990

Photochemical modification of lac repressor--III. Mutant I12X86 versus wild-type repressor.

Photodestruction of the two tryptophan (TRP) residues of the core of the wild-type Escherichia coli lac repressor has already been used as a probe in the study of interactions of the repressor with DNA and effectors. The good correlation between phenomena occurring in the core (photodestruction of TRP residues, effectors binding) and at the headpieces (DNA specific and non-specific binding) can be understood in terms of allosteric behavior of the protein. In the present study, the same approach is applied to a repressor with peculiar binding properties, the I12X86 mutant. The photodestruction of TRP residues of this tight binding repressor, bearing two different amino acids as compared to the wild-type one (Ser 61----Leu, Pro 3----Tyr) indicates a probably subtle (since not detected by classical spectroscopic methods) difference of structure of the entire protein and confirms the similarity between specific and non-specific binding of this mutant repressor to DNA, observed by other methods.

Escherichia coli↗

PubMed1990

Evidence for extended maintenance of the corpus luteum by uterine infusion of a recombinant trophoblast alpha-interferon (trophoblastin) in sheep.

Ovine trophoblastin (oTP) is a natural interferon of the class-II interferon-alpha subfamily. Recombinant ovine trophoblastin (r.oTP), produced by genetic engineering, was purified by anion-exchange HPLC. The product exhibited a high degree of homogeneity (greater than 98%), and similar immunological cross reaction and antiviral activity to natural oTP. Antiluteolytic activity of r.oTP was established by intrauterine injection in two groups of cyclic recipient ewes. Control group A included 10 ewes which received sterile BSA in saline twice daily for 8 days (from day 10-12 of oestrous cycle). Experimental group B included 17 ewes which received 80 micrograms (4 ewes), 170 micrograms (8 ewes) or 340 micrograms (5 ewes) r.oTP daily for 8 days. Maintenance of functional corpora lutea for 1 month or more was observed in 4 out of 5 ewes which received high doses of r.oTP. These results indicate that oTP alone extends luteal secretory activity.

Animals↗

PubMed1989

Cloning and expression of cDNA encoding ovine trophoblastin: its identity with a class-II alpha interferon.

The cDNAs encoding ovine trophoblastin (oTP) were isolated from an ovine embryo cDNA lambda gt 11 library by screening with a synthetic 29-mer oligodeoxynucleotide corresponding to amino acid (aa) residues 34 to 43 of oTP. The cDNA contained an open reading frame of 595 bp and the deduced amino acid sequence indicates a protein precursor of 195 aa. Nucleotide and amino acid sequence comparisons establish that oTP shares extensive homology with alpha-interferon (IFN-alpha) but is more closely related to the IFN-alpha sII subfamily. When the oTP cDNA was cloned into an eukaryotic expression vector and transfected in monkey COS cells, a high level of antiviral activity was detected. RNA blot analyses of total RNA reveal that the oTP-coding gene is expressed during a relatively short period (eleven to 21 days). The abundant expression of oTP mRNA corresponds closely to the time at which the embryo acts to extend luteal lifespan. RNAs homologous to oTP were also detected in goat and cow embryos at equivalent periods of their development, but not in the pig.

Amino Acid Sequence↗

PubMed1989

Limitation of DNA-4',6-diamidine-2-phenylindole assay in the presence of an excess of tRNA.

4',6-Diamidino-2-phenylindole, DAPI, forms fluorescent complexes with DNA. This property has been used to quantify DNA on the basis of fluorometric test. However, the fluorescence quantum yield of DAPI increases also with tRNA. DNA estimation needs particular care in the presence of tRNA. For DNA containing 50% adenine-thymine (AT), DAPI can be used if DNA represents at least 3.4% of the total nucleotide material. This percentage varies with the AT/guanosine-cytosine content. When the fraction of DNA decreases further, the DAPI assay can no longer be used.

DNA, Bacterial↗

PubMed1988

High homology between a trophoblastic protein (trophoblastin) isolated from ovine embryo and alpha-interferons.

Ovine trophoblastic protein B (oTPB), an embryonic protein, is a 20 kDa secretory protein which is synthesized by the ovine conceptus from days 12 to 22 of pregnancy. oTPB was purified by HPLC using ion-exchange chromatography on a DEAE column and was subsequently chromatographed on a reversed-phase column. Automated Edman degradation was then used to determine the N-terminal amino acid sequence up to 45 residues. The sequence data reveal a significant homology between oTPB and bovine interferons alpha of class II: 64% of the amino acids are identical and 75% are homologous. A highly conserved region including residues 23-44 exhibits 82% homology. Identity between oTPB and either HuIFN-alpha.9 or MuIFN alpha.1 is 55%. These alignments between oTPB and IFNs occur at the N-terminus of the mature proteins and proceed without deletion. These results suggest that oTPB is an embryonic interferon.

Amino Acid Sequence↗

PubMed1988

[Trophoblastic proteins].

Many studies conducted on human or animal placenta (chorion) suggest that the trophoblast is not only a passive filter between maternal and foetal blood flow, but is also endowed with complex functions. Factors of trophoblast origin involved in the mechanism of pregnancy recognition or maintenance of the progesterone environment required for the embryo survival, are reviewed. The main proteins involved in pregnancy are reported in table 1. Emphasis is laid on early signals of pregnancy which are of practical interest in human clinical medicine and animal husbandry. Among them, human chorionic gonadotropin (hCG), protein SP1 ("Schwangerschaftsprotein" 1) and some pregnancy-associated plasma proteins such as the PAPP A are very useful in the diagnosis of pregnancy, abortion, foetal abnormality or tumor in the human species. The presence of trophoblastin (presently studied in our laboratory) of a pregnancy-specific protein B and of early pregnancy factor (EPF) attest the establishment of pregnancy in domestic animals and in other mammals. The biological properties of some hormones such as placental lactogens (PL) or chorionic somatomammotropins (CS), human placental growth hormone (hPGH) contribute to a better understanding of the gestation function. Many other factors participate in the foetal development, for example, proliferin. Some proteins can display immunosuppressive properties or be responsible for the immune tolerance between the mother and the foetus. Although many placental proteins have already been defined, their biological functions have not yet been elucidated.

Animals↗

PubMed1987

Lac repressor-Lac operator complexes. Solution X-ray scattering and electrophoretic studies.

Complexes between the Lac repressor and a small DNA operator fragment (29 base pairs) were investigated using polyacrylamide gel electrophoresis and solution X-ray scattering. Titration of the DNA fragment with the repressor, followed by gel electrophoresis showed that only two types of complexes are formed with repressor/operator ratios of 0.5 and 2. Radii of gyration and forward scattered intensities were obtained from Guinier plots for repressor/operator ratios ranging from 0.3 to 2. They demonstrated that the first complex contains one repressor and two operators, whereas the second one contains four repressors and two operators. Mixing operator and repressor in equimolar concentrations leads to a mixture of both complexes. A possible model for the four repressor/two operator complex is proposed.

Base Sequence↗