Biomedical subjects

M Charlier

Publications and source records attributed to M Charlier.

At least 37 records · Page 2Linked to original sources

PubMed1998

Developmental expression and localization of the prolactin receptor (PRL-R) gene in ewe mammary gland during pregnancy and lactation: estimation of the ratio of the two forms of PRL-R messenger ribonucleic acid.

In this study, we have analyzed the developmental expression of the prolactin receptor (PRL-R) gene in the ewe mammary gland during pregnancy and lactation. Using Northern and slot-blot analysis and in situ hybridization, we showed that the level of PRL-R mRNA in mammary epithelial cells increased during the second half of pregnancy, decreased at the end of pregnancy, and remained relatively stable during lactation with a level above that observed at the beginning of pregnancy. As shown by RNase protection assay, the ratio of the long to the short form of the PRL-R mRNA was always above 1. This ratio increased between Day 70 of pregnancy and term and decreased progressively during lactation. The high level of PRL-R mRNA before the induction of alphaS1-casein gene expression suggests that PRL may be involved in the growth and development of the mammary gland. More precisely, the increase of the ratio of the long to the short form of the PRL-R during lactogenesis suggests that the latter form may have a dominant negative action in the activation of milk protein gene transcription. Thus the long/short-form ratio of the PRL-R may play a key role in the shift between growth and differentiation of the mammary gland.

Animals↗

PubMed1997

Recent developments and potentialities for reducing embryo mortality in ruminants: the role of IFN-tau and other cytokines in early pregnancy.

This review considers the potential reduction of embryo mortality in vitro and in vivo in ruminants. Data on cytokines provided by different fields of reproductive immunology and biology were collated. Because of the crucial importance of the local interactions between the embryo and its dam, the expression of growth-factor and cytokine genes was analysed in the embryo proper, trophoblast, oviduct and endometrium by reverse transcriptase polymerase chain reaction in sheep and in cattle during the pre- and periimplantation periods. Many deleterious cytokines, such as tumour necrosis factor-alpha, interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and beneficial cytokines, such as transforming growth factor-beta, leukaemia inhibiting factor, colony-stimulating factor-1 (CSF-1), granulocyte-macrophage CSF, IL-1, IL-3, IL-4, IL-6, IL-10 and IFN-tau appeared to be involved in embryo survival in ruminants and other species. Their administration is efficient in a murine experimental model (CBA/J x DBA/2) of embryonic and fetal mortality. For instance, recombinant ovine IFN-tau (roIFN-tau) injected at the moment of implantation drastically reduces embryonic mortality in this model. In ruminants, roIFN-tau and recombinant bovine IFN-tau are very efficient in maintaining progesterone luteal secretion in cyclic animals. The involvement of IFN-tau in the mechanisms of maternal pregnancy recognition are particularly detailed in relation to inhibition of 13,14 dihydro-15-keto-prostaglandin F2 alpha (PGFM) pulses and oxytocin uterine receptivity. A synthetic model of the anti-luteolytic effects of IFN-tau on the endometrial cell is proposed. Finally, the particular potential of serum pregnancy-specific proteins (PSPs: PSPB, PSP60, pregnancy-associated glycoprotein) for monitoring embryo survival, with examples given for cattle and sheep is underlined.

Amino Acid Sequence↗

PubMed1997

Sequence-dependent variations of DNA structure modulate radiation-induced strand breakage.

Using a 80 base pair DNA fragment, the sequence-dependence was compared for: (i) the probability of fast neutrons induced strand breakage, (ii) the accessibility of the H4'- and H5'-atoms to OH. attack, (iii) the width of the minor groove, and (iv) the probability of OH. reactions with H4'- or H5'-atoms. The probability of strand breakage was measured using sequencing gel electrophoresis. The accessibility and the probability of reaction were calculated for the energy-minimized modelled DNA fragment. A Monte-Carlo simulation was used for calculating the probabilities of H-atom abstraction by OH.. It was observed that reduced breakage occurs in sequences exhibiting low accessibility of H4' and H5'2 and low probability of H-atom abstraction by OH., due to a narrow, minor groove. This shows that the breakage probability at a given nucleotide site is not determined by the chemical nature of the nucleotide (A, T, G or C), but mainly by the local sequence-modulated intrinsic structure. Fitting the experimental results with the calculated probabilities of reaction suggests that a C4'-centered radical evolves towards a strand break three times more efficiently than the C5' one, and that half of the breaks occur via the 4'-path and half via the 5'-path.

Base Sequence↗

PubMed1997

Thiol WR-1065 and disulphide WR-33278, two metabolites of the drug ethyol (WR-2721), protect DNA against fast neutron-induced strand breakage.

The main metabolites of the cytoprotective drug Ethyol (Amifostine, WR-2721) are the thiol WR-1065 and the disulphide WR-33278 (formed by the oxidation of WR-1065). Both metabolites are well-known protectors against DNA damage induced by gamma-rays. Using supercoiled plasmid DNA and restriction fragments we show that they protect efficiently also in the case of fast neutrons. In anoxic conditions WR-1065 (Z = +2) protects by scavenging of OH. and by 'chemical repair' (by H donation from its SH function). WR-33278 (Z = +4) protects by scavenging of OH. and, in the case of the supercoiled plasmid DNA, by reducing the accessibility of radiolytic attack sites via the induction of packaging of DNA in liquid-crystalline condensates (observed by circular dichroism). Because of this second mechanism, the plasmid DNA is more efficiently protected by WR-33278 than by WR-1065, at concentration ratios > 1 drug/4 nucleotides. Moreover, using sequencing gel electrophoresis of irradiated fragments of known sequence, we show that the protection by the two metabolites is non-homogeneously distributed along the DNA sequence, with 'hot spots' of protection and with unprotected regions. Based on presented molecular modelling results we explain the sequence dependence of radioprotection by structural variations induced by the binding of the drugs.

Amifostine↗

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↗