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F De Lucia

Publications and source records attributed to F De Lucia.

14 recordsLinked to original sources
PubMed2000

Nucleosome dynamics. VI. Histone tail regulation of tetrasome chiral transition. A relaxation study of tetrasomes on DNA minicircles.

We have recently described the relaxation of mononucleosomes on an homologous series of 351-366 bp DNA minicircles, as a tool to study nucleosome structure and dynamics in vitro. Nucleosomes were found to have a tail-regulated access to three distinct DNA conformations, depending on the crossing between the entering and exiting DNAs, and its polarity. This approach was now used to explore tetrasome chiral transition, and the influence of the histone tails. The data confirmed the existence of two states, with linking number differences DeltaLk(t)=-0.74(+/-0.01) and +0.51(+/-0.06). As expected, the particle free energy is higher in the right-handed state (DeltaG(t)=1.9(+/-0.I) kT), but it decreased (to 1.3(+/-0.1) kT) upon histone acetylation and the addition of phosphate, a potent tail destabilizer. Removal of the tails with trypsin further decreased DeltaG(t) (to 0.6 kT), and also induced a loss of supercoiling in both states, to DeltaLk(t)=-0.64(+/-0.03) and +0. 35(+/-0.05). The loop end-conditions, and hence the parameters of the DNA superhelix, were then calculated for both states using the explicit solutions to the equations of the mechanical equilibrium in the theory of elastic rod model for DNA. Whereas the pitch of the DNA superhelix may be approximately equal and opposite in the two conformations, its radius (r) was 20% larger in the right-handed conformation, confirming previous observations by electron microscopy of a tetrasome lateral opening in that conformation. The above supercoiling losses were found to reflect a further 3 % increase in r (to 23 %) upon removal of the tails in the right-handed conformation, and a 14 % increase in the left-handed conformation. The use of composite tetramers with one histone tail intact and the other removed showed these effects to be essentially due to the H3 tails. Altogether, these results show that the H3 tails oppose the tetrasome opening which is expected to be required to relieve the clash between the entering and exiting DNAs in the course of the transition, but which also appears to be intrinsic to the protein reorientation mechanism. We propose that the block against opening results from the H3 tails intercalating into the small groove of the double helix at +/-10 bp from the dyad, and acting as wedges against local DNA straightening. The tails (especially H3) may therefore regulate tetrasome chiral transition in vivo.

Acetylation↗
PubMed1999

Nucleosome dynamics. II. High flexibility of nucleosome entering and exiting DNAs to positive crossing. An ethidium bromide fluorescence study of mononucleosomes on DNA minicircles.

H2A-H2B exchange with the intranuclear histone pool upon chromatin transcription in vivo is generally viewed as being triggered by the DNA positive supercoiling wave pushed by the elongating polymerase. This notion was tested here by investigating a potential release of H2A-H2B by ethidium bromide-induced positive supercoiling in the loop of mononucleosomes reconstituted on DNA minicircles. The results of gel electrophoresis, fluorescence titration and electron microscopy showed that such a positive supercoiling was not able to release H2A-H2B, nor to unfold the nucleosome to any detectable extent. The reason appeared to be the ease with which the loop could undergo a positive crossing, a surprising observation in view of the DNA left-handed wrapping around the octamer. Moreover, the influence of histone acetylation suggested that such loop flexibility to positive crossing is mediated by histone N-terminal tails which, by interacting with entering and exiting DNAs, reduce their electrostatic repulsion. These conclusions are confirmed and extended in the accompanying article through relaxation with topoisomerase I.

Animals↗
PubMed1999

Nucleosome dynamics. III. Histone tail-dependent fluctuation of nucleosomes between open and closed DNA conformations. Implications for chromatin dynamics and the linking number paradox. A relaxation study of mononucleosomes on DNA minicircles.

The mean linking number ( ) of the topoisomer equilibrium distribution obtained upon relaxation of DNA minicircles with topoisomerase I did not increase linearly, but rather in a step wise fashion, with DNA size between 351 and 366 bp. As a consequence, the corresponding linking number difference ( ) did not remain equal to 0, but rather oscillated between +/-0.3 with the periodicity of the double helix. This oscillation, not observed with plasmid-size DNA, is an expected consequence of the stiffness of short DNA. When minicircles were reconstituted with a nucleosome, the associated oscillated between approximately -1.4 +/-0. 2. This oscillation appears to result from the combined effects of DNA stiffness, and nucleosome ability to thermally fluctuate between three distinct DNA conformational states. Two of these states, a closed approximately 1.75-turn DNA conformation with negatively crossed entering and exiting DNAs, and an open approximately 1.4-turn conformation with uncrossed DNAs, are well known, whereas the third state, with a closed DNA conformation and DNAs tending to cross positively rather than negatively, is less familiar. Access to both closed "negative" and "positive" states appears to be mediated by histone N-terminal tails, as shown by specific alterations to the oscillation caused by histone acetylation and phosphate ions, a potent tail destabilizator. These results extend previous observations of ethidium bromide fluorescence titration in the accompanying article, which have pointed to an histone tail-dependent flexibility of entering and exiting DNAs to positive crossing. They also show that DNA wrapping around the histones occurred without twist alteration compared to the DNA free in solution, and reveal an intriguing new facet of the "linking-number-paradox" problem: the possibility for linkers in chromatin to adopt different crossing status within an overall dynamic equilibrium which may be regulated by histone acetylation.

Acetylation↗
PubMed1999

In vitro poly(ADP-ribosyl)ated histones H1a and H1t modulate rat testis chromatin condensation differently.

Rat testis H1 proteins were poly(ADP-ribosyl)ated in vitro. The modifying product, poly(ADP-ribose), was found covalently bound to each histone variant at various extents and exhibited distinct structural features (linear and short, rather than branched and long chains). Interest was focused on the somatic H1a, particularly abundant in the testis, as compared with other tissues, and the testis-specific H1t, which appears only at the pachytene spermatocyte stage of germ cell development. These H1s were modified with poly(ADP-ribose) by means of two in vitro experimental approaches. In the first system, each variant was incubated with purified rat testis poly(ADP-ribose)polymerase in the presence of [(32)P] NAD. In parallel, poly(ADP-ribosyl)ated H1s were also prepared following incubation of intact rat testis nuclei with [(32)P] NAD. In both experiments, the poly(ADP-ribosyl)ated proteins were purified from the native forms by means of phenyl boronic agarose chromatography. The results from both analyses were in agreement and showed qualitative differences with regard to the poly(ADP-ribose) covalently associated with H1a and H1t. Comparison of the bound polymers clearly indicated that the oligomers associated with H1a were within 10-12 units long, whereas longer chains (</=20 ADP-R units) were linked to H1t. Individual poly(ADP-ribosyl)ated H1s were complexed with homologous H1-depleted oligonucleosomes (0.5-2.5 kbp) in order to measure their ability to condensate chromatin, in comparison with the native ones. Circular dichroism showed that the negative charges of the oligomeric polyanion, although present in limited numbers, highly influenced the DNA-binding properties of the analyzed H1s. In particular, the poly(ADP-ribosyl)ated H1a and H1t had opposite effects on the condensation of H1-depleted oligonucleosomes.

Animals↗
PubMed1997

ADPribosylation reaction by free ADPribose in Sulfolobus solfataricus, a thermophilic archaeon.

In the archaeon Sulfolobus solfataricus, protein ADPribosylation by free ADPribose was demonstrated by testing both [adenine-14C(U)]ADPR and [adenine-14C(U)]NAD as substrates. The occurrence of this process was shown by using specific experimental conditions. Increasing the incubation time and lowering the pH of the reaction mixture enhanced the protein glycation by free ADPribose. At pH 7.5 and 10 min incubation, the incorporation of free ADPribose into proteins was highly reduced. Under these conditions, the autoradiographic pattern showed that, among the targets of ADPribose electrophoresed after incubation with 32P-NAD, the proteins modified by free 32P-ADPribose mostly corresponded to high molecular mass components. Among the compounds known to inhibit the eukaryotic poly-ADPribose polymerase, only ZnCl2 highly reduced the ADPribose incorporation from NAD into the ammonium sulphate precipitate. A 20% inhibition was measured in the presence of nicotinamide or 3-aminobenzamide. No inhibition was observed replacing NAD with ADPR as substrate.

Adenosine Diphosphate Ribose↗
PubMed1996

Poly(ADPribosyl)ation system in transcriptionally active rat testis chromatin fractions.

The rat testis chromatin fractions (soluble, S, and insoluble, P) were prepared by mild digestion of nuclei with DNAase I. They appeared to be different in specific biochemical features such as their transcriptional competence and protein patterns, the latter indicating according to results previously obtained, that the testis-specific H1t is preferentially associated to the soluble fraction, whereas the other H1 variants are localized in the pellet. S and P chromatins also differed in the distribution of the poly(ADP-ribosyl)ating system, (poly(ADP-ribose)polymerase, reaction product and acceptor proteins), detected by incubating nuclei with 32P-NAD. The 32P-modified H1s and core histones of both fractions, known as specific ADPribose target proteins, were separated by high performance liquid chromatography and it was demonstrated that the H1 variants from S and P are differently ADPribosylated, being H1t always the best acceptor, and that most of the ADPribosylated variants were solubilized after DNase I treatment. The further digestion of P chromatin with the nuclease produced a fraction (pP) devoid of most DNA, but particularly enriched in transcriptionally competent tracts. The low DNA content of pP chromatin, which reflects the typical feature of a nuclear matrix, corresponded to a relevant poly(ADPribosyl)ation, the highest as compared to S and P fractions. Moreover, long and branched chains of poly(ADP-ribose) were found associated to pP sample which resemble the products determined in the soluble chromatin.

Animals↗
PubMed1996

Interaction of the histone (H3-H4)2 tetramer of the nucleosome with positively supercoiled DNA minicircles: Potential flipping of the protein from a left- to a right-handed superhelical form.

We have studied the ability of the histone (H3-H4)2 tetramer, the central part of the nucleosome of eukaryotic chromatin, to form particles on DNA minicircles of negative and positive superhelicities, and the effect of relaxing these particles with topoisomerase I. The results show that even modest positive torsional stress from the DNA, and in particular that generated by DNA thermal fluctuations, can trigger a major, reversible change in the conformation of the particle. Neither a large excess of naked DNA, nor a crosslink between the two H3s prevented the transition from one form to the other. This suggested that during the transition, the histones neither dissociated from the DNA nor were even significantly reshuffled. Moreover, the particles reconstituted on negatively and positively supercoiled minicircles look similar under electron microscopy. These data agree best with a transition involving a switch of the wrapped DNA from a left- to a right-handed superhelix. It is further proposed, based on the left-handed overall superhelical conformation of the tetramer within the octamer [Arents, G., Burlingame, R. W., Wang, B. C., Love, W. E. & Moudrianakis, E. N. (1991) Proc. Natl.Acad. Sci. USA 88, 10148-10152] that this change in DNA topology is mediated by a similar change in the topology of the tetramer itself, which may occur through a rotation (or a localized deformation) of the two H3-H4 dimers about their H3-H3 interface. Potential implications of this model for nucleosome dynamics in vivo are discussed.

Animals↗
PubMed1995

ADP-ribosylation reactions in Sulfolobus solfataricus, a thermoacidophilic archaeon.

An ADP-ribosylating system was detected in a crude homogenate from Sulfolobus solfataricus, a thermophilic archaeon, optimally growing at 87 degrees C. The archaeal ADP-ribosylation reaction was time-, temperature- and NAD-dependent. It proved to be highly thermostable, with about 30% decrease of 14C incorporation from [14C]NAD on incubation at 80 degrees C for up to 24 h. The main reaction product was found to be mono-ADP-ribose. Testing both [adenine-14C(U)]NAD and [adenine-14C(U)]ADPR as substrates, it was found that acceptor proteins were modified by ADP-ribose both enzymatically, via ADP-ribosylating enzymes, and via chemical attachment of free ADP-ribose, likely produced by NAD glycohydrolase activity. The synthesis of ADP-ribose-protein complexes was shown to involve mainly acceptors with molecular masses in the 40-100 kDa range, as determined by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate.

Adenosine Diphosphate↗
PubMed1994

Histone-induced condensation of rat testis chromatin: testis-specific H1t versus somatic H1 variants.

Due the likely role of H1 histone variants in inducing the formation of folded DNA filaments with different stabilities, the condensing capacity of the testis-specific H1t versus the somatic variants was tested. Circular dichroism analyses of rat testis H1-depleted oligonucleosomes (5-2kbp) revealed that H1t, which appears in germ cells during the meiotic prophase of mammalian spermatogenesis, exerts the lowest condensing effect as compared to the other variants. The distribution of H1 subtypes among different chromatin fractions was also investigated and gave evidence that H1t is more abundant in chromatin regions which are more sensitive to DNAase I digestion.

Animals↗
PubMed1993

Effect of ADP-ribosylation reaction on bull testis chromatin.

Polynucleosomes prepared from bull testis nuclei were characterized: DNA length, by agarose gel electrophoresis, and distribution of poly(ADP-ribose)polymerase activity, by incubation with 0.64mM NAD were determined. Maximal activity was found in nucleosome fractions of 3-5 units. Chromatin fragments (5-2 kbp polynucleosomes) were analysed by circular dichroism in both native and ADP-ribosylated forms. The spectrum of the endogenously ADP-ribosylated polynucleosomes, compared to the native fraction, exhibited a higher ellipticity value and a shift of the cross-over point towards the lowest wavelengths, behaving like an H1-depleted chromatin.

Adenosine Diphosphate Ribose↗
PubMed1991

Antiinflammatory-antioxidant treatment with a methane sulfonanilide in allergen-induced asthma.

Two groups of six asthmatic patients with biphasic bronchospastic response to inhaled Dermatophagoides pteronyssinus allergen extract were studied in a double-blind fashion. Early and late asthmatic reactions to allergen inhalation challenge were determined before and at the end of a 2-week treatment period with nimesulide (100 mg bid orally), a sulfonanilide with antioxidant properties, or placebo. Bronchial responsiveness to methacholine was evaluated 24 hours before and after allergen inhalation challenges. The dose of allergen causing EAR (15% decrease in FEV1) and the severity of LAR (maximum FEV1 fall) were similar before and at the end of the treatment period in both groups. In patients treated with nimesulide, bronchial responsiveness to methacholine was significantly increased after allergen inhalation challenge both before and at the end of the treatment period. These results do not support the hypothesis that the production of oxygen-free radicals plays a significant role in the development of bronchial hyperresponsiveness and late phase reaction to allergen in asthma.

Adolescent↗
PubMedDate not supplied

Microcomputer-based coronary care unit central station.

A four-bed central station that can be connected to any commercial intensive-care bedside monitor was developed. The system is based on a personal computer (IBM-AT compatible) as a local unit and on a microcontroller Intel 8031 as a remote unit. Four ECG signals are low-pass filtered, multiplexed, sampled at 256-Hz per channel, 8-bit A/D converted, preprocessed, and converted to a serial format RS-232 by the remote unit. The real-time display of the signals is at the standard speed of 25 and 50 mm/sec. Heartrate, alarms, trend plots, and general patient data are shown on an Olivetti M280 and EGA 13'' color monitor as the local unit. The communication speed was set at 57.6 Kbaud full duplex. Additionally, to reach standard monitoring sweep rates using a 13'' screen with 640 x 350 pixels, an ECG data-compression algorithm was implemented in the remote unit. This unit can support up to eight input channels and can work with any personal computer, via RS-232, with the appropriate software. It also allows other signal preprocessing software that could be developed, such as QRS detection or ST segment quantification, to be loaded into its random access memory and to be run under PC command. The development of this system demonstrated the use of a widespread piece of commercial equipment, the PC, in a very specific application, CCU monitoring, assuring low-cost system implementation. This feature is particularly attractive in upgrading existing CCU units in less developed countries.

Biomedical Engineering↗