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Biomedical subjects

C Marion

Publications and source records attributed to C Marion.

At least 19 recordsLinked to original sources
PubMed2001

Tenascin-C aptamers are generated using tumor cells and purified protein.

Tenascin-C (TN-C) is an extracellular matrix protein that is overexpressed during tissue remodeling processes, including tumor growth. To identify an aptamer for testing as a tumor-selective ligand, SELEX (systematic evolution of ligands by exponential enrichment) procedures were performed using both TN-C and TN-C-expressing U251 glioblastoma cells. The different selection techniques yielded TN-C aptamers that are related in sequence. In addition, a crossover procedure that switched from tumor cell to purified protein selections was effective in isolating two high-affinity TN-C aptamers. When targeting tumor cells in vitro, the observed propensity of naive oligonucleotide pools to evolve TN-C aptamers may be due to the abundance of this protein. In vivo, TN-C abundance may also be well suited for aptamer accumulation in the tumor milieu. A size-minimized and nuclease-stabilized aptamer, TTA1, binds to the fibrinogen-like domain of TN-C with an equilibrium dissociation constant (K(d)) of 5 x 10(-9) m. At 13 kDa, this aptamer is intermediate in size between peptides and single chain antibody fragments, both of which are superior to antibodies for tumor targeting because of their smaller size. TTA1 defines a new class of ligands that are intended for targeted delivery of radioisotopes or chemical agents to diseased tissues.

Animals↗
PubMed1996

In vitro antimalarial activity of vegetal extracts used in West African traditional medicine.

Among strategies for the development of new antimalarials, a study of plants traditionally used in Africa against malaria has been pursued. Extracts obtained from the plants Azadirachta indica, Cinnamonum camphora, Lippia multiflora, Vernonia colorata, Guiera senegalensis, Combretum micranthum, and Ximenia americana, commonly used in Cote d'Ivoire by native healers for the treatment of malaria, were tested on two strains of Plasmodium falciparum: FcB1-Colombia (chloroquine-resistant) and F32-Tanzania (chloroquine-sensitive). Extracts were obtained after infusion and decoction, both techniques being used by most native healers. The antimalarial activities of the extracts were tested first by parasite 3H-hypoxanthine incorporation and second by visual evaluation of the activities of plant extracts on thin blood smears, which also permitted the determination of parasitic stages and parasite alteration. Among the seven plants tested, some had an apparent inhibitory effect on P. falciparum growth in vitro, while other seemed to be less efficient.

Animals↗
PubMed1995

Composition and antimalarial activity in vitro of volatile components of Lippia multiflora.

The essential oil of Lippia multiflora was prepared by hydrodistillation of leaves and stalks and characterized by GC and mass spectroscopy. The oil was tested for antimalarial activity on in vitro cultures of Plasmodium falciparum (FcB1-Columbia chloroquine-resistant strain and F32-Tanzania chloroquine-sensitive strain). The dilutions inhibiting the in vitro growth of the parasite by 50% 24 and 72 hr after administration of the essential oil to the parasite culture were 1/12,000 and 1/21,000, respectively. When tested on a highly synchronized culture, the essential oil inhibited growth mostly at the trophozoite-schizont step, indicating a potential effect on the first nuclear division of the parasite.

Animals↗
PubMed1993

Structural analysis of Trypanosoma brucei brucei chromatin by limited proteolysis. An electrical-birefringence study.

The sensitive electric-birefringence method was used to reveal structural differences between the soluble chromatin of procyclic Trypanosoma brucei brucei and the chromatin of the higher eukaryotes. The orientation of the nucleosomal chains and the presence of extended DNA were analysed from the sign and amplitude of the steady-state birefringence, and the conformational properties (overall dimensions and flexibility) were studied in relation to the orientational relaxation times. In contrast to the higher eukaryotes, the birefringence of T. brucei brucei is negative and of low amplitude, corresponding to that of H1-depleted rat liver nucleosomes. Furthermore, the relaxation times are very small, about 10 microseconds. If salt is added, the birefringence as well as the relaxation time decreases dramatically, indicating that condensation affects T. brucei brucei chromatin although it behaves like nucleosome filaments, with less stable DNA-protein interaction than for the higher eukaryotes. However, this condensation does not induce the formation of regular higher-order structure. This complies with the hypothesis that typical histone H1 is absent from T. brucei brucei chromatin and that a protein or protein domain fulfils the role of histone H1. The accessibility and structural role of histone-like proteins in T. brucei brucei chromatin were also investigated using limited proteolysis with enzymes covalently bound to nylon spheres. The analysis of protein products obtained after digestion with immobilized trypsin and subtilisin shows that proteins a and d, which are classified as H3 and H4 histones, respectively, are the first to be attacked. The changes in chromatin conformation indicate that chromatin undergoes a structural transition, leading to decondensation, as indicated by increases in negative birefringence and relaxation time, and to a change in its orientation mechanism, indicated by the appearance of a permanent moment. This result is very interesting since, in rat liver, H4 was very resistant and was the last histone to be attacked, suggesting internal location and its involvement in nucleosome stabilization rather than higher-order condensation. Therefore, in T. brucei brucei chromatin, the characteristic properties of proteins a and d (their composition and interaction with DNA), as well as their external location on the nucleosome surface, suggest that if these proteins play a role similar to that played by H3 and H4 in higher eukaryotes, probably through their N-terminal regions and interaction either with DNA or protein domains, the mechanisms involved in chromatin compaction are quite different.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

DNase activity of micrococcal endonuclease covalently immobilized on nylon and polystyrene.

Water-insoluble nucleases were prepared by immobilizing the endonuclease from S. aureus onto the surface of nylon-66 and polystyrene spheres. The activation phase of the synthetic supports was optimized to define optimal conditions of pH, temperature, and Ca2+ concentration for using immobilized enzymes. The activity, evaluated by hydrolysis of high-molecular-weight and supercoiled DNA, indicates that both derivatives are highly stable for storage and further use. Immobilization of the enzyme is much more effective when the covalent binding is performed on polystyrene. By using different activation methods with these matrices, a set of immobilized nucleases with various levels of enzymatic activity can be prepared. The possibility of working in a wide range of enzymatic activity and at low temperature and Ca2+ concentrations in different buffers makes these immobilized nucleases very useful for investigating accessible DNA regions in chromatin structure.

Biotechnology↗
PubMed1992

Structural differences between the chromatin of procyclic Trypanosoma brucei brucei and of higher eukaryotes as probed by immobilized trypsin.

Soluble chromatin of Trypanosoma brucei brucei procyclic culture forms was submitted to digestion with free or immobilized trypsin. Digestion with trypsin in salt solutions of low and high ionic strengths generated characteristic sets of limit histone peptides. After incubation of chromatin with immobilized trypsin in a solution of low ionic strength, histones were not degraded, whereas a selective proteolysis occurred at 50 mM NaCl. Histones a and d, which correspond to H3 and H4 of higher eukaryotes, were rapidly attacked. Histones b and c, the counterparts of H2A and H2B, were more resistant. The results indicated that probably the basic N-terminal tails of the proteins a and d are located on the surface of the core particle. The location of d on the surface differs from the internal one proposed for histone H4. The salt-induced increase of susceptibility of histones to proteolysis reflects structural changes of T.b. brucei chromatin, which may result in partial chromatin compaction.

Animals↗
PubMed1992

Caffeic glycoside esters from Jasminum nudiflorum and some related species.

Poliumoside, forsythoside B, echinacoside and arenarioside, caffeic glycoside esters, were isolated from several species of Oleaceae. The poliumoside/forsythoside ratio distinguishes Jasminum nudiflorum from J. mesnyi. Arenarioside and forsythoside B act in Forsythia species as good hybridization markers.

Caffeic Acids↗
PubMed1990

Use of an immobilized enzyme and specific antibodies to analyse the accessibility and role of histone tails in chromatin structure.

Using limited proteolysis with subtilisin bound to collagen membranes, the degradation of the histone proteins revealed by specific antibodies was correlated to changes in chromatin conformation and condensation monitored by circular dichroism and electric birefringence. This new approach allows us to detect for the first time a hierarchy of histone tails cleavages. The terminal domains of H1, the NH2-terminal tail of H3 and the carboxy-terminal ends of histones H2A and H2B were found to be cleaved already at the early stages of proteolysis and this led to a decondensation of polynucleosomal chains. Thereafter the C-terminal part of H3 and both NH2-terminal regions of H2A and H2B became rapidly cleaved, resulting in relative reorientation of swinging nucleosomes or partially unfolded segments. Unexpectedly, this removal of tails of H1, H2B, H2A and H3 is not accompanied by significant changes in DNA-protein interactions resulting in free-oriented DNA. This might suggest that histone-histone interactions play a central role in stabilizing the solenoid.

Animals↗
PubMed1990

Enzyme coupling method on calibrated nylon spheres: application to the selective trypsinization of histones in chromatin.

A new method consisting of a two-step activation was developed in order to covalently immobilize enzymes on calibrate nylon 66 spheres. This efficient method associates for the first time peptide bond cleavage and O-alkylation of the support. Optimal conditions for activation and protein coupling were defined, and immobilized trypsin was used to investigate the histone accessibility on chromatin. This approach, which allows us to degrade first progressively H1, indicates that H4 seems inaccessible both in relaxed and condensed chromatin.

Chemical Phenomena↗
PubMed1990

Linear contour length dependence of electrical polarizability of nucleosomal DNA fragments: implications for the flexibility of DNA.

The transient electric birefringence of monodisperse oligonucleosomal DNA ranging from 145 to 990 base pairs has been studied. The orientation of fragments can be described in terms of an induced dipole moment with a small contribution of a permanent dipole. The electrical polarizability delta alpha was found to increase linearly with the DNA contour length. This unexpected dependence might result from a bent structure of DNA already considerable for very short segments. The observed delta alpha values agree with a segmental orientation of rigid subunits of length 13-18 nm as estimated in the elastic model of DNA with a kink angle of about 41 degrees.

Animals↗
PubMed1990

Irreversible changes occur in chromatin structure upon dissociation of histone H1.

The role of histone H1 in the actual interactions bringing about chromatin folding is investigated by studying the reversibility of its dissociation. H1 was dissociated by increase of the NaCl concentration and reassociated by dialysis, without removal from the dialysis bag. To scrutinize the fidelity of this stoichiometric form of chromatin reconstitution, we use circular dichroism, nuclease digestion, thermal denaturation and the sensitive electric birefringence method. No alteration of the repeat length and no nucleosomal sliding are observed upon the reassociation procedure. However, under all the different conditions investigated, the original value of the positive electric birefringence is never recovered, indicating an irreversible change of structure. CD and melting profiles confirm that DNA-protein interactions are modified, and orientational relaxation time measurements indicate that these structural perturbations affect the salt-induced transition of polynucleosomal fibers. The striking conclusion of these studies is that variations of ionic concentration are sufficient to induce irreversible structural alterations affecting the higher-order folding of chromatin. It is of interest that the only sample which exhibits behavior upon reassociation comparable to that of native chromatin is the one which experienced the fastest salt transitions. We suggest that these conformational changes arise from the unbinding to DNA of certain basic tails of histone(s), and that a competition for DNA binding locations exists upon the reassociation. These results are then additional arguments (Mazen, A., Hacques, M.F. and Marion, C.,J. Mol. Biol. 194, 741-745 (1987)), to suggest that dissociation of H1 might modify a direct interaction between basic tails of core histones and H1.

Binding, Competitive↗
PubMed1990

Accessibility and structural role of histone domains in chromatin. biophysical and immunochemical studies of progressive digestion with immobilized proteases.

The accessibility and role of histone regions in chromatin fibres were investigated using limited proteolysis with enzymes covalently bound to collagen membranes. The changes in chromatin conformation and condensation monitored by various biophysical methods, were correlated to the degradation of the histone proteins revealed by antibodies specific for histones and histone peptides. Upon digestion with trypsin and subtilisin, chromatin undergoes successive structural transitions. The cleavage of the C-terminal domains of H1, H2A and H2B, and of the N-terminal tail of H3 led to a decondensation of chromatin fibres, indicated by increases in electric birefringence and orientational relaxation times. It corresponds to a 15% increase in linear dimensions. The degradation of the other terminal regions of histones H3, H2A and H2B resulted in the appearance of hinge points between nucleosomes without alteration of the overall orientation of polynucleosome chains. Despite the loss of all the basic domains of H1, H3, H2A and H2B, no significant change in DNA-protein interactions occurred, suggesting that most of these protease-accessible regions interact weakly, if at all, with DNA in chromatin. Further proteolysis led to H4 degradation and other additional cleavages of H1, H2B and H3. This caused the relaxation of no more than 8% of the total DNA but resulted in changes in the ability of chromatin to condense at high ionic strength. More extensive digestion resulted in a total unravelling of nucleosomal chains which acquired properties similar to those of H1-depleted chromatin, although the globular part of H1 was still present. The data suggest that histone-histone interactions between H1 and core histone domains play a central role in stabilizing the chromatin fibres, and cuts in H3, H2A and H2B as well as H1, seem necessary for chromatin expansion. On the contrary, H4 might be involved in the stabilization of nucleosomes only.

Animals↗
PubMed1989

The effect of a novel inhibitor of aldehyde dehydrogenase on viral replication.

The effect of AMPAL (4-amino 4-methyl 2-pentyne 1-al), an inhibitor of aldehyde dehydrogenase, on adenovirus type 5 (Ad5) replication was studied. AMPAL at 2 x 10(-4) M clearly reduced the cytopathic effect on HeLa cells but had no effect on cell growth at this concentration. Viral adsorption, penetration and protein synthesis were not affected by adding AMPAL at 2 hr post infection. When viral DNA synthesized in the presence of AMPAL was investigated, no significant inhibition was observed on either synthesis or the physicochemical properties of the neosynthesized DNA. However, there was a 4-fold increase in the amount of condensed DNA. In addition, AMPAL inhibited intracellular viral production (40%) and brought about concomitant inhibition of virus release (70%) into the medium. The absence of a quantitative relationship between the inhibition of viral DNA synthesis on one hand and that of viral production on the other may imply that the antiviral effect of AMPAL is indirectly mediated by the action of the malondialdehyde which has accumulated on some, as not yet identified, membrane constituent.

Adenoviruses, Human↗
PubMed1989

Structural differences between active and inactive mammalian 60S ribosomal subunits. Circular dichroism and electric birefringence studies.

The structure and conformation of different active and inactive forms of the 60S rat liver ribosomal subunits have been analyzed by electric birefringence and circular dichroism. These studies show the following: 1) When a phosphate buffer is used instead of a triethanolamine buffer, there are major changes in RNA stacking, RNA-protein interactions, and particle orientation and conformation with no concomitant loss in ribosome activity. 2) The inactivated subunits by K(+)-depletion exhibit the same electro-optical and near-UV CD behaviour than the active subunits in phosphate buffer. 3) Inactivation by EDTA-treatment leads to drastic changes in RNA structure, RNA-protein interactions and subunit conformation; the 60S particles behave like free RNA, indicating the absence of any stabilization of rRNA by ribosomal proteins. 4) The inactivation of subunits by depletion of either monovalent or divalent cations is accompanied by a net decrease of the alpha-helicity of the ribosomal proteins. 5) The transition from active to inactive form of 60S subunits may involve protein modifications, likely dependent on a specific array of cations. 6) RNA has a certain degree of liberty within the subunits and one can suppose that this property is responsible for the flexible structure of ribosome.

Animals↗
PubMed1989

[Adsorbed plant extracts, use of extracts of dried seeds of Orthosiphon stamineus benth].

The work presented is related to the elaboration and the control of spray dried vegetable extracts of Orthosiphon Stamineus Benth. From these studies, it feasibility of the spray dried extracts. The best result concerning a pulverulent structure and the stability under moisture conditions is obtained using Aerosil 200. Pharmacological studies on rats have shown a diuretic activity under our experimental conditions with alcoholic and water extracts.

Diuretics↗
PubMed1988

Ribosomal proteins S2, S6, S10, S14, S15 and S25 are localized on the surface of mammalian 40 S subunits and stabilize their conformation. A study with immobilized trypsin.

Trypsin immobilized on collagen membranes has been used to digest accessible ribosomal proteins of rat liver 40 S subunits. Six proteins (S2, S6, S10, S14, S15 and S25) have been found to be highly exposed on the surface of 40 S particles. They appear to be in close physical contact and localized in the same region of the subunit, most likely protruding at its surface. Electric birefringence reveals that digestion of these proteins results in unfolding of subunits: the birefringence of 40 S particles becomes negative, like that of RNA, the relaxation time undergoes a 15-fold decrease and the mechanism of orientation is drastically modified.

Animals↗