Biomedical subjects

M Charlier

Publications and source records attributed to M Charlier.

At least 19 recordsLinked to original sources

PubMed1996

Comparative study of DNA radiolysis by fast neutrons and gamma-rays.

The effect of fast neutrons on cells is different from that of gamma-rays: the relative (to gamma) biological effect (RBE) is higher than one and the oxygen enhancement ratio (OER) is lower than that of gamma-rays. We searched for differences between the effects of the two radiations on DNA, the critical target of radiations. Using a model plasmid DNA we observed that for the same absorbed dose, fast neutrons induce twice fewer single strand breaks (SSB) and 1.5 more double strand breaks (DSB) than gamma-rays. A transition metal ion, the Cu++, is a better sensitizer of DNA breakage with fast neutrons than with gamma-rays. In anoxia, cysteamine, a positively charged thiol, is a better radioprotector against neutrons than against gamma-rays. In presence of cysteamine, the OER is lower for neutrons than for gamma-rays. These results are discussed in terms of different physical properties of the two types of radiation.

Copper↗

PubMed1996

Sequence-modulated radiosensitization of DNA by copper ions.

Plasmid DNA and restriction fragments of 80 and 120 base pairs were irradiated with fast neutrons in the presence of CuCl2. The number of single and double strand breaks is higher in the presence than in the absence of Cu2+ ions. The radiosensitizing effect was lower for solutions of high compared with low ionic strength, and also lower for deoxygenated than for aerated solutions. This effect was inhibited by EDTA, catalase and Tris, but not by ethanol. Superoxide dismutase partially inhibited the effect of low copper concentrations (< 1 Cu2+/nucleotide). Saturation of the solutions with N2O removed the effect for these concentrations of copper. The sensitization occurred preferentially at pyrimidines (thymines > cytosines) situated 5' to one or several purines (guanine > adenine) or located between two purines, at runs of purines (guanine > adenine), and at combinations of such sequences. The results can be only partially explained by a Fenton-like mechanism involving radiation induced hydrated electrons and hydrogen peroxide, which produces OH. radicals at the sites of binding of copper on DNA. The regions around these binding sites may undergo conformational changes. A second path for sensitization could be the enhancement of the efficiency of cleavage by the radiolytically produced OH. radicals in these conformationally modified regions.

Base Sequence↗

PubMed1996

DNA breakage upon K-shell excitation of phosphorus as a model for direct effects in radiation biology.

Single-strand breaks (SSBs) and double-strand breaks (DSBs) induced in DNA under phosphorus K-shell resonant absorption have been studied using supercoiled plasmids. The kinetics of the production of SSBs and DSBs exhibits a linear and a quadratic dependence, respectively, on photon fluence. Cross sections and quantum yields have been measured. The resonant photoexcitation of the phosphorus atoms was found to increase the DSB/SSB ratio compared to the off-resonance excitation. This enhancement factor can be related to the measured enhancement of the rate of cellular death and gene mutation in yeast under similar experimental conditions reported previously in the literature. Such resonant excitation of a specific atom belonging to DNA turns out to be an elegant method to investigate pure direct effects.

DNA Damage↗

PubMed1995

Calculation of hydroxyl radical attack on different forms of DNA.

A stochastic model of hydroxyl radical reaction with the DNA macromolecule in dilute aqueous solution is presented. It is based on the Monte Carlo method, the model of Smoluchowski, the model of the regular DNA atomic structure and the knowledge of reaction rate constants for .OH radical reaction with DNA constituents. The calculated respective overall probability of .OH radical reaction with sugar or base moiety dependents on DNA form (A, B or Z), base composition (AT/GC content) and strandedness (single and double). While for all three DNA forms studied (A, B and Z) as well as for both single- and double-stranded B-DNA, the specific probability of .OH radical attack on nucleobases is modulated by their sequence, the attack on deoxyriboses is only slightly sequence dependent (except for Z-DNA). The model predictions are compared to the experimental patterns of strand breaks and alkali revealed breaks for single and double stranded DNA in B-form and discussed in terms of molecular mechanisms of DNA damage induced by hydroxyl radical.

Base Composition↗

PubMed1995

Radiation-induced damages in single- and double-stranded DNA.

In the present study, we searched for possible effects of DNA strandedness (single and double), on two types of damages, frank strand breaks (FSB, observed at neutral pH) and alkali labile sites (ALS, leading to breaks at alkaline pH) induced by irradiation with gamma-rays (60Co) or fast neutrons (p34,Be). Sequencing gel electrophoresis allowed us to follow the occurrence of these damages at each nucleotide site in single (ss-ss), double (ds-ds), and half single-half double (ss-ds and ds-ss) stranded oligonucleotides. Globally, in DNA with random sequences of bases, no differences in FSB and ALS yield between the single and the double-stranded conformations were observed. One observes, however, an increased alkaline lability at some guanine sites belonging to single-stranded region of ss-ds or ds-ss. Nevertheless, strandedness influences the radiosensitivity of some particular sequences, i.e. the 5'-AATT sequences. This region is less radiosensitive than the rest of DNA in the double helical, but not in the single-stranded conformation. The results are discussed in terms of DNA conformation.

Base Sequence↗

PubMed1995

Radioprotection of DNA by polyamines.

Putrescine, spermidine and spermine are natural polyamines bearing at neutral pH the net electrical charges +2, +3 and +4 respectively. We report here the radioprotective effect of these polyamines on the radiolysis of pBR322 plasmid DNA. We observe a very efficient protection against fast neutron-induced single and double-strand breakage in the presence of spermine and spermidine, and a significantly less efficient protection in the presence of putrescine. An ionic strength dependence is observed for spermidine and spermine, but not for putrescine. Circular dichroism measurements show spermidine- and spermine-induced structural modifications of DNA, i.e. the formation of tightly packaged condensates in the concentration range corresponding to radioprotection. No structural change is observed for concentrations of putrescine affording radioprotection. We explain the radioprotection by: (1) the scavenging of OH radicals in the bulk, essentially observed in the case of putrescine; (2) a local scavenging at the sites of binding of polyamines; and (3) the reduced accessibility of the attack sites in the condensed structures induced by spermine or spermidine.

Biogenic Polyamines↗

PubMed1994

Radiolytic signature of Z-DNA.

Ionizing radiations induce various damages in DNA via the hydroxyl radical OH. generated by the radiolysis of water. We compare here the radiosensitivity of B- and Z-DNA, by using a Z-prone stretch included in a plasmid. In the supercoiled plasmid, the stretch is in the Z-form, whereas it is in the B-form when the plasmid is relaxed. Frank strand breaks (FSB) and alkali-revealed breaks (ARB) were located and quantified using sequencing gel electrophoresis. We show that B- and Z-DNA have the same mean sensitivity towards radiolytic attack, for both FSB and ARB. Nevertheless, the guanine sites are more sensitive, and the cytosine sites less sensitive in Z- than in B-DNA, leading to a characteristic signature of the Z-form. The comparison of experiments with the outcome of a Monte Carlo simulation of OH. radical attack suggests that transfer of initial damage from a guanine base to its attached sugar or the adjacent 3' cytosine is more important in Z-DNA than in B-DNA.

Base Sequence↗

PubMed1994

Identification of the DNA-interacting sites of proteins: microsequencing of the peptides cross-linked to 5-bromouracil substituted DNA.

Photochemical induced cross-links between protein and nucleic acids are useful tools in the study of the protein-DNA interactions. The substitution of thymine by 5-bromouracil in DNA increases the photocross-linking yield, and reduces the direct damages to both DNA and proteins. Using the lac repressor-DNA non-specific interaction system, we have developed a procedure to identify the interaction site on the protein. Sensitive, accurate and inexpensive in time and material, this procedure is based on the possibility of sequencing peptides in the presence of a large excess of DNA. The obtained result (the implication of His 29) agrees with previous work.

Amino Acid Sequence↗

PubMed1994

Sites of strand breakage in DNA irradiated by fast neutrons.

Therapeutic fast neutrons are densely ionizing particles, with a high relative biological effectiveness relative to 60Co gamma rays (RBE) and a low oxygen enhancement ratio (OER). The molecular basis of their properties is not yet entirely understood. In a previous work, we have shown that neutrons induce a different number of DNA frank strand breaks as compared to gamma photons, and we have revealed the presence of breaks due to the direct effects of neutrons. In the present work, we searched for eventual differences in the chemical nature of the attacked sites in DNA irradiated in oxygenated diluted solution. We compare our results with neutrons to those previously reported by other authors using gamma- or X-rays. Using sequencing gel electrophoresis of short natural DNA restriction fragments, or synthetic oligonucleotides, we have shown that, in the case of neutrons, the attack occurs with almost the same probability, at each nucleotide, as reported for gamma- and X-rays. The doubling of bands in the bottom of gels shows the presence of two types of termini, the 3'-phosphate and the 3'-phosphoglycolate. Upon neutron irradiation, the 3'-phosphate end appears with a higher yield than the 3'-phosphoglycolate, whereas equal amounts were obtained with gamma- or X-rays.

Base Sequence↗

PubMed1994

Radiosensitivity of DNA minicircles.

DNA minicircles of 207 bp were constructed by the ligation of linear restriction fragments in the presence of various concentrations of ethidium bromide. Three topoisomers characterized by linking numbers (Lk) of 20, 19 and 18, and with helical repeats of 10.35, 10.9 and 11.5 bp/turn respectively, were obtained. They are called, respectively, relaxed minicircle or topoisomer 0, topoisomer -1 and topoisomer -2. Owing to the limited flexibility of such small circles, the stress created by the lack of 1 or 2 turns cannot be eliminated by a spatial circle-axis writhing (supercoiling) of the circular molecules. These two undertwisted, stressed topoisomers have to adopt a flat, non-crossed shape, similar to that of the relaxed minicircle. The three minicircles were irradiated with gamma-rays or fast neutrons. The same yields of single-strand breaks, double-strand breaks and alkali-induced single-strand breaks were observed for the three topoisomers showing that their base and sugar moieties are attacked equally by gamma photon- or fast neutron-induced radicals. We conclude that untwisting of a B helix does not modify the radiosensitivity of DNA.

Base Sequence↗

PubMed1993

Radiolytic footprinting. Beta rays, gamma photons, and fast neutrons probe DNA-protein interactions.

Ionizing radiations induce numerous damages in DNA, especially strand breaks. The hydroxyl radical OH., produced by the radiolysis of water, is mainly responsible for this effect. The fact that strand breakage occurs at all nucleotides and that bound proteins may locally radioprotect DNA at the binding site lead us to develop a radiolytic footprinting method to study DNA-protein interactions. Three different radiations were used: beta rays, gamma photons, and fast neutrons. In order to validate this technique, three well-known interaction systems were tested: the lac repressor-lac operator of Escherichia coli, the cyclic AMP receptor protein (CRP) of E. coli and its specific site in the lac regulation region, and the core nucleosome. Radiolytic footprinting gives results similar to those obtained by more classical probes: DNase I, complexes of orthophenanthroline (OP) and copper, complexes of ethylenediaminetetraacetate ion (EDTA) and iron, and UV light. For the same system (lac repressor), irradiation with either gamma photons or fast neutrons gives identical results.

Animals↗

PubMed1993

Photo-cross-linking of CRP to nonspecific DNA in the absence of cAMP. DNA interacts with both the N- and C-terminal parts of the protein.

Adenosine cyclic 3',5'-phosphate receptor protein (CRP or CAP) is a regulatory protein involved in the transcription of several operons in Escherichia coli. cAMP-independent, nonspecific complexes of CRP and DNA were investigated by photochemical cross-linking of the protein to nonspecific DNA, whose thymines are substituted by 5-bromouracil (BrUra). The cross-linked protein was completely digested by trypsin, and the covalently bound peptides were sequenced. We identified two regions of the protein in close contact with DNA: one in the C-terminal part, overlapping the canonical helix-turn-helix motif, and the other one in the N-terminal part, which is usually not considered to belong to the DNA-interacting domain of CRP. This result lead us to propose models for nonspecific interaction, where the DNA is in contact with both the N- and C-terminal parts of the protein.

Amino Acid Sequence↗

PubMed1993

N-acetylcysteine and captopril protect DNA and cells against radiolysis by fast neutrons.

N-Acetylcysteine and captopril, respectively mucolytic and antihypertensive drugs, contain free sulfhydryl groups. Since in general thiols have well-established radioprotective abilities, we sought putative radioprotective effects of these drugs against therapeutic fast neutrons. We show that pBR322 plasmid DNA is indeed protected against radiolytic strand breakage by both drugs. The oxygen independent protection is consistent with a hydroxyl radical scavenging mechanism. A clonogenicity assay reveals an increase of the survival of SCL-1 cultured keratinocytes irradiated in the presence of the drugs compared with cells irradiated without drugs. Our results suggest possible interferences between treatment with drugs bearing-SH groups and radiotherapy.

Acetylcysteine↗

PubMed1993

Radioprotection of DNA by a DNA-binding protein: MC1 chromosomal protein from the archaebacterium Methanosarcina sp. CHTI55.

The archaebacterial chromosomal protein MC1 binds tightly and unspecifically to DNA; binding protects DNA against radiolysis by fast neutrons. At low covering of pBR322 plasmid DNA, one bound protein protects some 50 attack sites (phosphate-sugar moieties) against both single (ssb) and double strand breaks (dsb). At high covering of plasmid, protection against dsb becomes almost complete, although about half of the attack sites remain accessible to ssb. DNA restriction fragments were used to investigate the organization of the complexes, and its consequences on DNA radiolysis. Sequencing gel electrophoresis of the radiolytically-broken fragments are almost regular in the absence of protein, showing that breakage occurs at every base. In the presence of the protein, a periodic protection pattern is observed. The period of 11 base pairs is interpreted as the minimum distance between two adjacent MC1 proteins.

Amino Acid Sequence↗

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↗