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J Bednar

Publications and source records attributed to J Bednar.

At least 19 recordsLinked to original sources
PubMed2006

Pulling the chromatin.

Nucleosome is the basic subunit of the chromatin, which organizes the genomic DNA within the cell nucleus. It was understood in the last decade that beside the DNA compaction it plays an important role in the regulation of the gene expression. In its intact form, the nucleosome represents an important mechanical barrier and, among others, it prevents access to the DNA and blocks the transcription elongation. Therefore, it has become important to know the forces and energies necessary to destabilize the nucleosome in order to understand the DNA-related processes. Stretching the chromatin fibre using micromanipulation techniques (e.g. optical tweezers) is an ideal approach to study the nucleosomal stability and the parameters that can modify it. In this short review we will discuss the existing data and potential difficulties that this state-of-the-art technique still has to overcome.

Chromatin↗
PubMed2005

Microbiological characterization of winery effluents: an inventory of the sites for different treatment systems.

In a more and more regulated and socially pressured environment, the durable management of winery effluents must take into account their characteristics and their potential impact on their natural setting. The object of this exploratory study is to establish an inventory of the microbiological composition of winery effluents coming from different treatment systems. We have observed that winery effluents are charged with micro-organisms, by a factor that ranges from 10(5) to 10(8) UFC/ml, and that the level of "microbiological pollution" is independent of the type of system. The composition of the flora is closely tied to the time of year and therefore to winery activities, so certain micro-organisms will be favoured in certain periods and others will have a tendency to decrease. We have seen that from one year to another our observations remain identical; the flora equilibrium therefore occurs systematically and naturally. Faecal germs are found in very small quantities in winery effluent treatment systems. They represent minor sanitary risks. Good correlations were observed between some micro-organisms and some physical-chemical parameters (COD). It is, however, difficult to use these "easy-to-measure" parameters as reliable markers of certain microbial populations.

Agriculture↗
PubMed1999

MENT, a heterochromatin protein that mediates higher order chromatin folding, is a new serpin family member.

Terminal cell differentiation is correlated with the extensive sequestering of previously active genes into compact transcriptionally inert heterochromatin. In vertebrate blood cells, these changes can be traced to the accumulation of a developmentally regulated heterochromatin protein, MENT. Cryoelectron microscopy of chicken granulocyte chromatin, which is highly enriched with MENT, reveals exceptionally compact polynucleosomes, which maintain a level of higher order folding above that imposed by linker histones. The amino acid sequence of MENT reveals a close structural relationship with serpins, a large family of proteins known for their ability to undergo dramatic conformational transitions. Conservation of the "hinge region" consensus in MENT indicates that this ability is retained by the protein. MENT is distinguished from the other serpins by being a basic protein, containing several positively charged surface clusters, which are likely to be involved in ionic interactions with DNA. One of the positively charged domains bears a significant similarity to the chromatin binding region of nuclear lamina proteins and with the A.T-rich DNA-binding motif, which may account for the targeting of MENT to peripheral heterochromatin. MENT ectopically expressed in a mammalian cell line is transported into nuclei and is associated with intranuclear foci of condensed chromatin.

Amino Acid Sequence↗
PubMed1999

The nature of the nucleosomal barrier to transcription: direct observation of paused intermediates by electron cryomicroscopy.

Transcribing SP6 RNA polymerase was arrested at unique positions in the nucleosome core, and the complexes were analyzed using biochemical methods and electron cryomicroscopy. As the polymerase enters the nucleosome, it disrupts DNA-histone interactions behind and up to approximately 20 bp ahead of the elongation complex. After the polymerase proceeds 30-40 bp into the nucleosome, two intermediates are observed. In one, only the DNA ahead of the polymerase reassociates with the octamer. In the other, DNA both ahead of and behind the enzyme reassociates. These intermediates present a barrier to elongation. When the polymerase approaches the nucleosome dyad, it displaces the octamer, which is transferred to promoter-proximal DNA.

Binding Sites↗
PubMed1998

Nucleosomes, linker DNA, and linker histone form a unique structural motif that directs the higher-order folding and compaction of chromatin.

The compaction level of arrays of nucleosomes may be understood in terms of the balance between the self-repulsion of DNA (principally linker DNA) and countering factors including the ionic strength and composition of the medium, the highly basic N termini of the core histones, and linker histones. However, the structural principles that come into play during the transition from a loose chain of nucleosomes to a compact 30-nm chromatin fiber have been difficult to establish, and the arrangement of nucleosomes and linker DNA in condensed chromatin fibers has never been fully resolved. Based on images of the solution conformation of native chromatin and fully defined chromatin arrays obtained by electron cryomicroscopy, we report a linker histone-dependent architectural motif beyond the level of the nucleosome core particle that takes the form of a stem-like organization of the entering and exiting linker DNA segments. DNA completes approximately 1.7 turns on the histone octamer in the presence and absence of linker histone. When linker histone is present, the two linker DNA segments become juxtaposed approximately 8 nm from the nucleosome center and remain apposed for 3-5 nm before diverging. We propose that this stem motif directs the arrangement of nucleosomes and linker DNA within the chromatin fiber, establishing a unique three-dimensional zigzag folding pattern that is conserved during compaction. Such an arrangement with peripherally arranged nucleosomes and internal linker DNA segments is fully consistent with observations in intact nuclei and also allows dramatic changes in compaction level to occur without a concomitant change in topology.

Animals↗
PubMed1998

Linker histones stabilize the intrinsic salt-dependent folding of nucleosomal arrays: mechanistic ramifications for higher-order chromatin folding.

Defined nucleosomal arrays reconstituted from core histone octamers and twelve 208 bp tandem repeats of Lytechinus 5S rDNA (208-12 nucleosomal arrays) possess the ability to form an unstable folded species in MgCl2 whose extent of compaction equals that of canonical higher-order 30 nm diameter chromatin structures [Schwarz, P. M., and Hansen, J. C. (1994) J. Biol. Chem. 269, 16284-16289]. To address the mechanistic functions of linker histones in chromatin condensation, purified histone H5 has been assembled with 208-12 nucleosomal arrays in 50 mM NaCl. Novel purification procedures subsequently were developed that yielded preparations of 208-12 chromatin model systems in which a majority of the sample contained both one histone octamer per 5S rDNA repeat and one molecule of histone H5 per histone octamer. The integrity of the purified 208-12 chromatin has been extensively characterized under low-salt conditions using analytical ultracentrifugation, quantitative agarose gel electrophoresis, electron cryomicroscopy, and nuclease digestion. Results indicate that histone H5 binding to 208-12 nucleosomal arrays constrains the entering and exiting linker DNA in a way that produces structures that are indistinguishable from native chicken erythrocyte chromatin. Folding experiments performed in NaC1 and MgC12 have shown that H5 binding markedly stabilizes both the intermediate and extensively folded states of nucleosomal arrays without fundamentally altering the intrinsic nucleosomal array folding pathway. These results provide new insight into the mechanism of chromatin folding by demonstrating for the first time that distinctly different macromolecular determinants are required for formation and stabilization of higher-order chromatin structures.

Animals↗
PubMed1998

Dosimetry during the first IBIS facility flight.

The dosimetry of cosmic rays was performed during the first experimental flight of the IBIS facility. Different thermoluminescent detectors (TLD) have been used to measure the contribution of the low linear energy transfer component (LET < 10 keV/micrometer) and plastic nuclear track detectors (PNTD) for the high linear energy tranfer (LET) component. Several parameters of tracks have been measured to determine the LET spectra of primary and secondary charged particles. The total absorbed dose rate (TLD+PNTD) during the flight was 0.23 mGy/day and the dose equivalent rate using the ICRP 60 was 0.52 mSv/day. The corresponding mean quality factor was 2.4. These results are in agreement with those obtained aboard the MIR station with a tissue equivalent proportional counter.

Cosmic Radiation↗
PubMed1997

Opposite effect of counterions on the persistence length of nicked and non-nicked DNA.

Using cryo-electron microscopy we reconstructed the three-dimensional trajectories adopted in cryovitrified solutions by double-stranded DNA molecules in which the backbone of one strand lacked a phosphate at regular intervals of 20 nucleotides. The shape of such nicked DNA molecules was compared with that of DNA molecules with exactly the same sequence but without any single-stranded scissions. Upon changing the salt concentration we observed opposite effects of charge neutralization on nicked and non-nicked DNA. In low salt solutions (10 mM Tris-HCl, 10 mM NaCl) the applied dense nicking caused ca 3.5-fold reduction of the DNA persistence length as compared with non-nicked DNA. Upon increasing the salt concentration (to 150 mM NaCl and 10 mM MgCl2) the persistence length of non-nicked DNA appreciably decreased while that of nicked DNA molecules increased by a factor of 2.

DNA↗
PubMed1996

Ex-vivo and in-vivo platelet function in patients with severe hypercholesterolemia undergoing LDL-apheresis.

Patients with severe familial hypercholesterolemia (HC) show abnormal platelet function and shortened platelet survival. Atherosclerosis is associated with platelet hyperactivity. Low-density lipoporotein (LDL)-apheresis eliminates the most atherogenic lipid fraction and inhibits the progression of atherosclerosis inducing even regression. In order to assess the influence of LDL-apheresis on platelet function ex-vivo and in-vivo, 6 patients with severe heterozygous HC, all of them being pharmacologically treated with HMG-CoA reductase inhibitors and anion exchange resins were investigated. Ex-vivo platelet function was assessed by the aggregation response to ADP before starting apheresis treatment, as well as after 2 and 24 weeks, respectively. In-vivo platelet function was determined by measuring platelet survival after radiolabeling with 111In-oxine before starting LDL-apheresis and after 24 weeks of twice monthly treatment. LDL-apheresis therapy induced a significant (p < 0.01) drop in cholesterol by 64%, LDL-cholesterol by 77% and in triglycerides by 46% over a period of 24 weeks. ADP-induced platelet aggregation revealed a decreased aggregability of platelets with a decline in the maximal amplitude and the slope of the response curve. Changes in platelet sensitivity to prostaglandins (PG) were significantly for PGI2, but did not reach statistical significance for PGE1. The results revealed a significant (p < 0.001) increase in platelet survival of 111In-oxine-radiolabeled autologous platelets from a mean of 106.50 hours before to 137.50 hours (p < 0.01) after treatment, being accompanied by an increase in labeling efficiency (p < 0.001) and recovery (p < 0.001). These data provide evidence for improved hemostatic regulation in vivo as a result of maintainance of lipid-lowering achieved with LDL-apheresis.

Adult↗
PubMed1995

Determination of DNA persistence length by cryo-electron microscopy. Separation of the static and dynamic contributions to the apparent persistence length of DNA.

Axial deflection of DNA molecules in solution results from thermal motion and intrinsic curvature related to the DNA sequence. In order to measure directly the contribution of thermal motion we constructed intrinsically straight DNA molecules and measured their persistence length by cryo-electron microscopy. The persistence length of such intrinsically straight DNA molecules suspended in thin layers of cryo-vitrified solutions is about 80 nm. In order to test our experimental approach, we measured the apparent persistence length of DNA molecules with natural "random" sequences. The result of about 45 nm is consistent with the generally accepted value of the apparent persistence length of natural DNA sequences. By comparing the apparent persistence length to intrinsically straight DNA with that of natural DNA, it is possible to determine both the dynamic and the static contributions to the apparent persistence length.

Base Sequence↗
PubMed1995

Chromatin conformation and salt-induced compaction: three-dimensional structural information from cryoelectron microscopy.

Cryoelectron microscopy has been used to examine the three-dimensional (3-D) conformation of small oligonucleosomes from chicken erythrocyte nuclei after vitrification in solutions of differing ionic strength. From tilt pairs of micrographs, the 3-D location and orientation of the nucleosomal disks, and the paths of segments of exposed linker can be obtained. In "low-salt" conditions (5 mM NaCl, 1 mM EDTA, pH 7.5), the average trinucleosome assumes the shape of an equilateral triangle, with nucleosomes at the vertices, and a length of exposed linker DNA between consecutive nucleosomes equivalent to approximately 46 bp. The two linker DNA segments converge at the central nucleosome. Removal of histones H1 and H5 results in a much more variable trinucleosome morphology, and the two linker DNA segments usually join the central nucleosome at different locations. Trinucleosomes vitrified in 20 mM NaCl, 1 mM EDTA, (the salt concentration producing the maximal increase in sedimentation), reveal that compaction occurs by a reduction in the included angle made by the linker DNA segments at the central nucleosome, and does not involve a reduction in the distance between consecutive nucleosomes. Frequently, there is also a change in morphology at the linker entry-exit site. At 40 mM NaCl, there is no further change in trinucleosome morphology, but polynucleosomes are appreciably more compact. Nevertheless, the 3-D zig-zag conformation observed in polynucleosomes at low salt is retained at 40 mM NaCl, and individual nucleosome disks remain separated from each other. There is no evidence for the formation of solenoidal arrangements within polynucleosomes. Comparison of the solution conformation of individual oligonucleosomes with data from physical measurements on bulk chromatin samples suggests that the latter should be reinterpreted. The new data support the concept of an irregular zig-zag chromatin conformation in solution over a range of ionic strengths, in agreement with other in situ (McDowall, A.W., J.M. Smith, and J. Dubochet. 1986, EMBO (Eur. Mol. Biol. Organ.) J.5: 1395-1402; Horowitz, R.A., D.A. Agard, J.W. Sedat, and C.L. Woodcock, 1994. J. Cell Biol. 125:1-10), and in vitro conclusions (van Holde, K., and J. Zlatanova. 1995. J. Biol. Chem. 270:8373-8376). Cryoelectron microscopy also provides a way to determine the 3-D conformation of naturally occurring chromatins in which precise nucleosome positioning plays a role in transcriptional regulation.

Animals↗
PubMed1994

The twist, writhe and overall shape of supercoiled DNA change during counterion-induced transition from a loosely to a tightly interwound superhelix. Possible implications for DNA structure in vivo.

A cryo-electron microscopy study of supercoiled DNA molecules freely suspended in cryo-vitrified buffer was combined with Monte Carlo simulations and gel electrophoretic analysis to investigate the role of intersegmental electrostatic repulsion in determining the shape of supercoiled DNA molecules. It is demonstrated here that a decrease of DNA-DNA repulsion by increasing concentrations of counterions causes a higher fraction of the linking number deficit to be partitioned into writhe. When counterions reach concentrations likely to be present under in vivo conditions, naturally supercoiled plasmids adopt a tightly interwound conformation. In these tightly supercoiled DNA molecules the opposing segments of interwound superhelix seem to directly contact each other. This form of supercoiling, where two DNA helices interact laterally, may represent an important functional state of DNA. In the particular case of supercoiled minicircles (178 bp) the delta Lk = -2 topoisomers undergo a sharp structural transition from almost planar circles in low salt buffers to strongly writhed "figure-eight" conformations in buffers containing neutralizing concentrations of counterions. Possible implications of this observed structural transition in DNA are discussed.

DNA, Superhelical↗
PubMed1991

Japanese NiTi coils used to move molars distally.

Japanese NiTi superelastic coils, exerting 100 gm of force, were compressed against the maxillary first molars and moved the molars distally 1 to 1.5 mm/month. Anchorage was obtained with a modified Nance appliance cemented onto the first premolars in conjunction with a fixed appliance.

Bicuspid↗