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J C Hansen

Publications and source records attributed to J C Hansen.

At least 55 records · Page 3Linked to original sources
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

Enhanced transcription factor access to arrays of histone H3/H4 tetramer.DNA complexes in vitro: implications for replication and transcription.

Defined model systems consisting of physiologically spaced arrays of H3/H4 tetramer.5S rDNA complexes have been assembled in vitro from pure components. Analytical hydrodynamic and electrophoretic studies have revealed that the structural features of H3/H4 tetramer arrays closely resemble those of naked DNA. The reptation in agarose gels of H3/H4 tetramer arrays is essentially indistinguishable from naked DNA, the gel-free mobility of H3/H4 tetramer arrays relative to naked DNA is reduced by only 6% compared with 20% for nucleosomal arrays, and H3/H4 tetramer arrays are incapable of folding under ionic conditions where nucleosomal arrays are extensively folded. We further show that the cognate binding sites for transcription factor TFIIIA are significantly more accessible when the rDNA is complexed with H3/H4 tetramers than with histone octamers. These results suggest that the processes of DNA replication and transcription have evolved to exploit the unique structural properties of H3/H4 tetramer arrays.

Animals↗
PubMed1998

Mechanism of Rab geranylgeranylation: formation of the catalytic ternary complex.

Rab proteins are geranylgeranylated on one or two C-terminal cysteines by Rab geranylgeranyl transferase (RabGGTase). The reaction is dependent on a Rab-binding protein, termed Rab escort protein (REP). Here, we studied the role of REP in the geranylgeranylation reaction. We first characterized the interaction between REP and ungeranylgeranylated Rab using analytical ultracentrifugation and a fluorescence-based assay. We measured an equilibrium dissociation constant of 0.2 microM for the formation of a 1:1 REP-Rab complex and showed that this interaction relies mostly on ionic bonds and does not involve the two C-terminal cysteine residues. Second, we show that REP is required for recognition of Rab by RabGGTase and therefore that the REP-Rab complex is the true substrate for RabGGTase. Third, we show that free REP inhibits the geranylgeranylation reaction, suggesting that the complex is recognized by RabGGTase primarily via a REP-binding site. Our data suggest a model whereby REP behaves kinetically as an essential activator of the reaction.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Elements in autopsy liver tissue samples from Greenlandic Inuit and Danes. I. Sulphur, chlorine, potassium and bromine measured by X-ray fluorescence spectrometry.

The purpose of this study was to measure the content of the elements Sulphur (S), Chlorine (C1), Potassium (K) and Bromine (Br) in normal liver tissue samples from Greenlandic Inuit using X-ray fluorescence spectrometry, and compare the results with those obtained in normal liver tissue samples from Danes. Liver tissue sample were obtained at autopsy from 50 Greenlandic Inuit (27 men, 23 women) with a median age of 61 years (range 20-83) and from 74 Danes (44 men, 30 women) with a median age of 52 years (range 15-87). In Inuit, the content of elements given as median and (5-95 percentile) was: sulphur, 108.07 mmol/kg dry liver (86.78 - 169.44); chlorine, 92.16 mmol/kg dry liver (45.39-128.42); potassium, 181.66 mmol/kg dry liver (146.41-236.35); bromine, 0.0901 mmol/kg dry liver (0.0563-0.1589). In Danes, the corresponding values were: sulphur, 147.58 mmol/kg dry liver (70.41-236.81); chlorine, 96.95 mmol/kg dry liver (54.01-162.52); potassium, 198.40 mmol/kg dry liver (150.68-256.37); bromine, 0.1101 mmol/kg dry liver (0.0701 - 0.4203). None of the elements displayed any significant gender difference, neither in Inuit nor in Danes. Inuit had a lower liver content of sulphur (p < 0.0001), potassium (p < 0.008) and bromine (p < 0.002) as compared with Danes.

Adult↗
PubMed1998

Disruption of higher-order folding by core histone acetylation dramatically enhances transcription of nucleosomal arrays by RNA polymerase III.

We have examined the effects of core histone acetylation on the transcriptional activity and higher-order folding of defined 12-mer nucleosomal arrays. Purified HeLa core histone octamers containing an average of 2, 6, or 12 acetates per octamer (8, 23, or 46% maximal site occupancy, respectively) were assembled onto a DNA template consisting of 12 tandem repeats of a 208-bp Lytechinus 5S rRNA gene fragment. Reconstituted nucleosomal arrays were transcribed in a Xenopus oocyte nuclear extract and analyzed by analytical hydrodynamic and electrophoretic approaches to determine the extent of array compaction. Results indicated that in buffer containing 5 mM free Mg2+ and 50 mM KCl, high levels of acetylation (12 acetates/octamer) completely inhibited higher-order folding and concurrently led to a 15-fold enhancement of transcription by RNA polymerase III. The molecular mechanisms underlying the acetylation effects on chromatin condensation were investigated by analyzing the ability of differentially acetylated nucleosomal arrays to fold and oligomerize. In MgCl2-containing buffer the folding of 12-mer nucleosomal arrays containing an average of two or six acetates per histone octamer was indistinguishable, while a level of 12 acetates per octamer completely disrupted the ability of nucleosomal arrays to form higher-order folded structures at all ionic conditions tested. In contrast, there was a linear relationship between the extent of histone octamer acetylation and the extent of disruption of Mg2+-dependent oligomerization. These results have yielded new insight into the molecular basis of acetylation effects on both transcription and higher-order compaction of nucleosomal arrays.

Acetylation↗
PubMed1998

Effects of dietary seal oil on fat metabolism.

In the 1970s, Bang and Dyerberg demonstrated that a high intake of n-3 acids in Greenland protected against ischemic heart disease. This started the interest in fish oil as a preventive component in cardiac disease. The fatty acid composition in Greenlandic diet is quite different from the one in the Danish diet, being lower in saturated fatty acids and polyunsaturated fatty acids (PUFAs) and higher in monounsatured fatty acids (MUFAs). During the last decade, evidence has suggested that the MUFA (18:1) is not a neutral dietary component but has a positive effect of its own. This paper reports on a current project undertaken to study the effect of marine MUFAs and PUFAs on atherosclerosis-related parameters and to evaluate their possible synergistic effect in the prevention of atherosclerosis. The project has recently started, so at the moment results cannot be presented. The study will be carried out as an intervention study on 50 healthy volunteers. Capsules of Greenlandic halibut oil (rich in MUFAs) and "Biomarin" (rich in n-3 PUFAs) will be tested against seal oil.

Adult↗
PubMed1998

The human health programme under AMAP. AMAP Human Health Group. Arctic Monitoring and Assessment Program.

The human health programme of the first phase of AMAP was planned at an international meeting held in Nuuk, Greenland, October 1992. As the most vulnerable period to adverse effects of contaminants is during fetal development, it was decided to concentrate on analyses of umbilical cord blood and maternal blood. The programme was designed as a core programme in which 150 sample pairs should be collected in each of the 8 arctic countries and analyzed for persistant organic pollutants (POPs) and heavy metals (mercury, lead and cadmium). As some essential elements such as copper, zinc and selenium interfere with heavy metal toxicity these elements should also be analyzed. Additional analyses such as nickel and arsenic in urine, mercury in hair, and POPs in breast milk could be incorporated regionally according to specific local conditions. Radionucleides were not a major focus in the human programme as this issue was be dealt with by AMAP's radiation group. Implementation of the programme was a problem in most of the countries due to lack of funding. However, an offer from Canada to analyze all contaminants in 50 samples from each country enabled the first comparative circumpolar study of human exposure to contaminants to be completed. The study confirmed that in general the most important source of exposure to both POPs and mercury is food of marine origin and that Greenlanders and Inuit from the Canadian Arctic, due to their traditional lifestyle, are among the most highly exposed populations in the Arctic. This is not a result of local pollution in Greenland and Canada, but is due to long range transport of persistent contaminants through the atmosphere and their biomagnification in the marine food chain. For these reasons the most important recommendation of the first AMAP assessment is that priority should be given to the expeditious completion of negotiations to establish protocols for the control of POPs and heavy metals under the Convention on Long Range Transboundary Air Pollution.

Arctic Regions↗
PubMed1997

Hybrid trypsinized nucleosomal arrays: identification of multiple functional roles of the H2A/H2B and H3/H4 N-termini in chromatin fiber compaction.

A defined 12-mer nucleosomal array in solution exists in a complex equilibrium between an unfolded 29S conformation, a 40S folding intermediate, an extensively folded 55S conformation, and soluble oligomeric states formed from cooperative intermolecular association of individual 12-mer arrays. Proteolytic removal of all of the core histone N-terminal tail domains previously has been shown to abolish both salt-dependent nucleosomal array folding and oligomerization. To elucidate the individual contributions of the H2A/H2B and H3/H4 tail domains to nucleosomal array condensation, "hybrid" trypsinized nucleosomal arrays have been assembled from tandemly repeated 5S rDNA and either trypsinized H3/H4 tetramers and intact H2A/H2B dimers or trypsinized H2A/H2B dimers and intact H3/H4 tetramers. Neither of the hybrid trypsinized arrays formed either the 40S or the 55S folded conformations in 2 mM MgCl2. In >/=4 mM MgCl2, however, both fully trypsinized arrays and each hybrid trypsinized array formed the 40S folding intermediate, but not the 55S conformation. In contrast to folding, each hybrid trypsinized nucleosomal array oligomerized completely in MgCl2. These studies have identified three mechanistically distinct functions performed by the core histone N-termini during salt-dependent condensation of nucleosomal arrays. The complexity of tail domain function in chromatin is discussed in the context of a competitive interaction model in which the core histone N-termini provide direct mechanistic links between the structure and function of the chromatin fiber.

Cations, Divalent↗
PubMed1997

Analytical ultracentrifugation and agarose gel electrophoresis as tools for studying chromatin folding in solution.

Analytical ultracentrifugation and agarose gel electrophoresis each can be used to accurately quantify changes in structure that accompany chromatin folding in solution. Analytical ultracentrifugation directly measures the extent of compaction of each species present in a chromatin sample under a wide range of solution conditions. Agarose gel electrophoresis yields information about changes in the average surface charge density, size and/or shape, and conformational flexibility during chromatin folding. When used together, these methodologies are particularly powerful. Protocols for the characterization of chromatin folding by analytical ultracentrifugation and agarose gel electrophoresis are described. Discussion focuses on analysis and interpretation of experimental chromatin folding data.

Chromatin↗
PubMed1997

Identification and interpretation of complexity in sedimentation velocity boundaries.

Synthetic sedimentation velocity boundaries were generated using finite-element solutions to the original and modified forms of the Lamm equation. Situations modeled included ideal single- and multicomponent samples, concentration-dependent samples, noninteracting multicomponent samples, and reversibly self-associating samples. Synthetic boundaries subsequently were analyzed using the method of van Holde and Weischet, and results were compared against known input parameters. Results indicate that this analytical method provides rigorous diagnostics for virtually every type of sample complexity encountered experimentally. Accordingly, both the power and utility of sedimentation velocity experiments have been significantly expanded.

Computer Simulation↗
PubMed1997

Influence of network topology on the elasticity of the red blood cell membrane skeleton.

A finite-element network model is used to investigate the influence of the topology of the red blood cell membrane skeleton on its macroscopic mechanical properties. Network topology is characterized by the number of spectrin oligomers per actin junction (phi a) and the number of spectrin dimers per self-association junction (phi s). If it is assumed that all associated spectrin is in tetrameric form, with six tetramers per actin junction (i.e., phi a = 6.0 and phi s = 2.0), then the topology of the skeleton may be modeled by a random Delaunay triangular network. Recent images of the RBC membrane skeleton suggest that the values for these topological parameters are in the range of 4.2 < phi a < 5.5 and 2.1 < phi s < 2.3. Model networks that simulate these realistic topologies exhibit values of the shear modulus that vary by more than an order of magnitude relative to triangular networks. This indicates that networks with relatively sparse nontriangular topologies may be needed to model the RBC membrane skeleton accurately. The model is also used to simulate skeletal alterations associated with hereditary spherocytosis and Southeast Asian ovalocytosis.

Actins↗
PubMed1997

Effect of toxaphene on estrogen receptor functions in human breast cancer cells.

Toxaphene (polychlorinated camphenes) is an insecticidal mixture of >670 chemicals, which was widely used until the mid 1980s. Due to their lipophilic and volatile nature, these chemicals accumulate in animal and human tissues and continue to be a major contaminant in marine and freshwater biota. Cytotoxic and genotoxic effects in mammalian test systems suggest that toxaphene is a carcinogen and reports support the hypothesis that toxaphene could have tumor-promoting potential in human breast tissue. In order to examine the potential of toxaphene as an environmental endocrine disrupter, we investigated its effect on the estrogen receptor (ER) function in human breast cancer MCF-7 cells. Using transient gene expression experiments, we observed approximately 60% and 80% inhibition of the constitutive and 17beta-estradiol induced ER-dependent transactivation, respectively. The involvement of the ER in the ability of toxaphene to block the estrogen action was verified by cotransfection studies in ER-negative MDA-MB-231 cells. The interference of toxaphene with the ER mediated responses was supported by a significant suppression of endogenously expressed pS2 RNA and decreased levels of secreted pS2 protein. These reproducible results indicate that toxaphene can disturb hormonal signals mediated by the ER and suggest that these environmental chemicals have potential endocrine disrupting activities which may affect the reproductive health and increase the risk of carcinogenesis.

Breast Neoplasms↗
PubMed1997

Dentinal and pulpal uptake of mercury from lined and unlined amalgam restorations in minipigs.

The aim of the present investigation was to examine dentin and pulp in amalgam-restored teeth in minipigs with respect to presence of mercury, and to evaluate whether lining of restored cavities with a resin-modified glass ionomer cement had any effect on the penetration of mercury. Class I and V cavities were cut in 49 posterior and canine teeth of 4 minipigs. 35 cavities were restored with amalgam and 14 with composite material. 26 of the amalgam cavities were lined with a resin-modified glass ionomer cement. Following observation periods of 17-28 months, the pigs were killed and the jaws sectioned. After decalcification, the teeth were processed for light or electron microscopy examinations. Mercury was visualized by autometallography. Silver-enhanced mercury was found in all teeth with amalgam, whereas teeth with composites were devoid of mercury. Mercury could be traced in the odontoblast processes, in the body of odontoblasts, and on rare occasions in the nerve tissue of the pulp from lined and from unlined amalgam restorations. The present study thus demonstrates transport of mercury through dentinal tubules to the pulp, and that lining of cavities with a resin-modified glass ionomer cement does not prevent penetration of mercury.

Animals↗
PubMed1996

Selective attention to stimulus location modulates the steady-state visual evoked potential.

Steady-state visual evoked potentials (SSVEPs) were recorded from the scalp of human subjects who were cued to attend to a rapid sequence of alphanumeric characters presented to one visual half-field while ignoring a concurrent sequence of characters in the opposite half-field. These two-character sequences were each superimposed upon a small square background that was flickered at a rate of 8.6 Hz in one half-field and 12 Hz in the other half-field. The amplitude of the frequency-coded SSVEP elicited by either of the task-irrelevant flickering backgrounds was significantly enlarged when attention was focused upon the character sequence at the same location. This amplitude enhancement with attention was most prominent over occipital-temporal scalp areas of the right cerebral hemisphere regardless of the visual field of stimulation. These findings indicate that the SSVEP reflects an enhancement of neural responses to all stimuli that fall within the "spotlight" of spatial attention, whether or not the stimuli are task-relevant. Recordings of the SSVEP provide a new approach for studying the neural mechanisms and functional properties of selective attention to multi-element visual displays.

Adult↗
PubMed1996

Reversible oligonucleosome self-association: dependence on divalent cations and core histone tail domains.

Regularly spaced nucleosomal arrays equilibrate between unfolded and highly folded conformations in <2 mM MgCl2, and self-associate above 2 mM MgCl2 [Schwarz, P. M., & Hansen, J. C. (1994) J. Biol. Chem. 269, 16284-16289]. Here we use analytical and differential sedimentation techniques to characterize the molecular mechanism and determinants of oligonucleosome self-association. Divalent cations induce self-association of intact nucleosomal arrays by binding to oligonucleosomal DNA and neutralizing its negative charge. Neither linker histones nor H2A/H2B dimers are required for Mg2+ - dependent self-association. However, divalent cations are unable to induce self-association of trypsinized nucleosomal arrays lacking their N- and C-terminal core histone tail domains. This suggests that the H3/H4 tail domains directly mediate oligonucleosome self-association through a non-Coulombic-based mechanism. Self-association occurs independently of whether the oligonucleosome monomers are folded or unfolded. The first step in the self-association pathway is strongly cooperative and produces a soluble association intermediate that sediments approximately 10 times faster than the oligonucleosome monomers. The size of the oligonucleosome polymers increases rapidly as a consequence of small increases in the divalent cation concentration, eventually producing polymeric species that sediment at >> 10 000 S. Importantly, all steps in the self-association pathway are freely reversible upon removal of the divalent cations. Taken together, these data indicate that short oligonucleosome fragments composed of only core histone octamers and DNA possess all of the structural features required to achieve chromosome-level DNA compaction. These findings provide a molecular basis for explaining many of the recently uncovered structural features of interphase and metaphase chromosomal fibers.

Animals↗