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The role of a microscopic colloidally stabilized phase in solubilizing oligoamine-condensed DNA complexes.

DNA complexes of spermine and spermidine become resolubilized at very high concentrations of the oligoamine. It has been postulated that high oligoamine concentrations shift the DNA from the globule back to the coil phase. The present study indicates that DNA resolubilization at high concentrations of spermine and spermidine is explained by formation of small particles of condensed DNA that cannot be precipitated by centrifugation. The fact that DNA stays condensed during resolubilization was confirmed using a relatively new condensation assay and three independent microscopic techniques. A considerable portion of DNA was found to be in particles with diameter <100 nm. Formation of such small particles is likely to be caused by colloidal forces. The ability to form small, condensed DNA particles in solutions that contain high concentrations of oligocation should aid in the design of synthetic DNA vectors for gene transfer and gene therapy and in the handling of DNA for diagnostic studies.

Amines↗

Condensation of supercoiled DNA induced by MnCl2.

Multivalent cations condense DNA in vitro, but it had been thought that a valence of at least + 3 was required in aqueous solution. We have found that Mn2+ can produce toroidal condensates of supercoiled plasmid DNA, but not of linearized plasmid. Mg2+ does not cause condensation, and neither MgCl2 nor NaCl can negate the effect of MnCl2, indicating that the condensation mechanism with Mn is not primarily electrostatic. Supercoiled MnDNA is more extensively digested than the linear form by S1 nuclease. Supercoiling appears to cooperate with Mn2+ in stabilizing helix distortions and also provides a "pressure" that enhances lateral association.

Chlorides↗

Evaluation of the counterion condensation theory of polyelectrolytes.

We compare free energies of counterion distributions in polyelectrolyte solutions predicted from the cylindrical Poisson-Boltzmann (PB) model and from the counterion condensation theories of Manning: CC1 (Manning, 1969a, b), which assumes an infinitely thin region of condensed counterions, and CC2 (Manning, 1977), which assumes a region of finite thickness. We consider rods of finite radius with the linear charge density of B-DNA in 1-1 valent and 2-2 valent salt solutions. We find that under all conditions considered here the free energy of the CC1 and the CC2 models is higher than that of the PB model. We argue that counterion condensation theory imposes nonphysical constraints and is, therefore, a poorer approximation to the underlying physics based on continuum dielectrics, point-charge small ions, Poisson electrostatics, and Boltzmann distributions. The errors in counterion condensation theory diminish with increasing distance from, or radius of, the polyion.

Biophysical Phenomena↗

Cell condensation in chondrogenic differentiation.

Reduction of intercellular spaces in the areas of prospective cartilage and bone formation (precartilage condensation) precedes chondrogenesis and may represent an important step in the process of cartilage differentiation during limb skeletogenesis. We have attempted to clarify the role of the microenvironment established during cell condensation, taking advantage of a tissue culture model system that allows condensation (i.e., increased cell density due to cell aggregation) and chondrogenic differentiation (i.e., synthesis of cartilage-specific extracellular matrix proteins, such as type II collagen and acquisition of a chondrocyte morphology) of chick embryo cartilage-derived undifferentiated cells. To prevent condensation cells were grown in carboxymethylcellulose and changes in the differentiation pathway were evaluated. In another series of experiments, we have separated single cells from the aggregated cells and analyzed their differentiation properties. Morphological analyses and the evaluation of type II collagen expression, at both the protein and the mRNA level, show that a reduced rate of cell clustering and cell to cell contact parallels a reduction of cell recruitment into the differentiation program. On the basis of our results, we suggest that the following cascade of events regulates the early stages of chondrocyte differentiation: (a) the acquisition of the ability to establish cell to cell contacts, (b) the formation of a permissive environment capable of activating the differentiation program, and (c) the expression of differentiation markers.

Animals↗

Observation of DNA-polymer condensate formation in real time at a molecular level.

Dynamic real time assembly of toroidal and rod-like DNA condensates has been visualised using atomic force microscopy. Imaging has been conducted in an aqueous environment allowing the visualisation of hydrated, pegylated-polymer DNA condensates undergoing dynamic structural movement and conformational change. A major hurdle in the field of gene delivery is cellular transfection and the subsequent transfer of condensed genetic material to the cell nucleus. An increased understanding of the process of DNA condensation will aid the development and optimisation of gene delivery vectors.

Cations↗

Preferential condensation of SAR-DNA by histone H1 and its SPKK containing octapeptide repeat motif.

Linker histone H1 binds preferentially the scaffold associated region (SAR) DNA elements that contain characteristic oligo dA x dT tracts. In the present study, we have compared the condensation brought about by histone H1 of a SAR DNA fragment in the histone spacer region of Drosophila melanogaster with that of a random DNA (pBR322 EcoRI-SalI) fragment by circular dichroism spectroscopy. The condensation of the SAR DNA fragment by histone H1 is 3-4-fold higher than that of the random DNA fragment. A 16-mer peptide, ATPKKSTKKTPKKAKK, the sequence that is present in the C-terminus of histone Hld, which has recently been shown to possess DNA and chromatin condensing properties, also condenses the SAR DNA fragment preferentially in a highly cooperative manner. We have proposed a model for the dynamics of chromatin structure involving histone H1-SAR DNA interaction through SPKK containing peptide motifs and its competition by AT-hook peptides present in the nonhistone chromosomal proteins like HMG-I and HMG-Y.

Amino Acid Sequence↗

Cationic silanes stabilize intermediates in DNA condensation.

In vitro condensation of DNA has been widely studied to gain insight into the mechanisms of DNA compaction in biological systems such as chromosomes and phage heads and has been used to produce nanostructured particles with novel material and functional properties. Here we report on the condensation of DNA in aqueous solutions by cationic silanes, which combine the condensing properties of polyamines with the cross-linking chemistry of silanes. DNA can be reversibly condensed into classical toroidal and rod-shaped structures with these agents. At low silane concentrations DNA forms a variety of looped structures with well-defined characteristics, including flower- and sausage-shaped forms. These structures suggest that at low silane concentrations a DNA-DNA contact in which the strands are at very large angles to each other is stabilized. Changes in these structures observed as a function of silane concentration suggest possible pathways for the formation of toroids and rods.

Cations↗

Prematurely condensed chromosomes and meiotic abnormalities in unfertilized human oocytes after ovarian stimulation with and without gonadotropin-releasing hormone agonist.

OBJECTIVE: To investigate the incidence of meiotic abnormalities, aneuploidy, and prematurely condensed sperm chromosomes in failed fertilized oocytes after controlled ovarian hyperstimulation (COH). DESIGN: Retrospective analysis of air-dried preparations of unfertilized oocytes. SETTING: University hospital-based infertility clinic. PATIENT(S): Thirty-three patients undergoing IVF having only tubal factor as the cause of infertility. Twelve patients (13 cycles) underwent treatment with hMG alone (-GnRH agonist [GnRH-a]), and 21 patients (24 cycles) underwent treatment with leuprolide acetate (LA) and hMG (+GnRH-a group). INTERVENTION(S): Standard IVF-ET treatment cycle for ovarian stimulation using hMG with or without LA. MAIN OUTCOME MEASURE(S): The meiotic stage, ploidy, and the presence of prematurely condensed sperm chromosomes were determined in 161 air-dried preparations of unfertilized oocytes. RESULT(S): Significantly more unfertilized oocytes were at metaphase II in the -GnRH-a group as compared with the +GnRH-a group, with significantly fewer exhibiting meiotic aberrations. Aneuploidy rates did not differ between groups. However, significantly more oocytes in the +GnRH-a group revealed prematurely condensed sperm chromosomes than in the -GnRH-a group. CONCLUSION(S): The use of GnRH-a for COH does not have an impact on aneuploidy rates in failed fertilized oocytes. However, the higher incidence of meiotic aberrations and prematurely condensed sperm chromosomes in the unfertilized population indicates that some retrieved oocytes exhibit incomplete nuclear and cytoplasmic maturation after the use of this agonist.

Adult↗

On the adsorption and condensed film formation of dodecyl-, tetradecyl-, hexadecyl-, and octadecyltrimethylammonium bromides at the mercury/electrolyte interface.

The adsorption and condensed film formation of dodecyl (DTAB)-, tetradecyl (TTAB)-, hexadecyl (CTAB)-, and octadecyl (OTAB)-trimethylammonium bromides on the hanging mercury electrode is studied in KBr as supporting electrolyte, at various temperatures from 5 to 45 degrees C. A condensed film is formed at negative potentials and at room temperature only in the presence of CTAB. The decrease of the temperature favors the formation of the condensed film. A transition temperature is observed for the film formation. Capacity-time curves at the potentials where the film is formed show a nucleation and growth mechanism, with induction time depending not only on the final potential but also on the initial potential range, although it is in the desorption region. In this temperature range no film is observed for DTAB and TTAB. However, the film is observed for OTAB, but only at higher temperatures, and is more easily formed with increasing temperature. The film is formed in a certain potential region and the nucleation rate increases while moving toward more negative potentials. Hysteresis phenomena are observed during changes of scan direction. The capacity vs time curves for OTAB, where condensed film is formed, are treated using an Avrami plot formulation and have been explained as progressive one-dimensional nucleation with a decrease of the nucleation rate during the overall film formation. The results show a marked effect of the chain length of the alkyl chain on the film formation.

Journal Article↗

Reactions of the four diastereomeric 16-amino-17-hydroxy-3-methoxy-estra-1,3,5(10)-trienes with aromatic ortho-hydroxy and heteroaromatic alpha-aldehydes and with 1, 3-dicarbonyl compounds-molecular structures of condensation products and of copper(II) complexes.

Vicinal amino alcohols of steroids have been used as starting materials for the synthesis of chiral ligands with defined arrangements of functional groups. Condensation of the four diastereomeric 16,17-steroid amino alcohols 1a-1d with aromatic o-hydroxy and heteroaromatic alpha-aldehydes afforded the Schiff bases 2-6. When the 16,17-substituted compounds 2d, 5d, 6a, and 6d were in solution, the isomeric oxazolidines were detectable by (1)H NMR spectroscopy. The formation of oxazolidines could be avoided by using bulky aldehydes. Reduction of the Schiff bases (also in mixtures with oxazolidines) with NaBH(4) yielded the new N-substituted amino alcohols 12-15. The condensation products of 1a-1d with 1,3-dicarbonyl compounds (7 and 8) exhibited the 1-enamino-3-oxo structure ((1)H NMR spectroscopy). By means of X-ray analysis of 2a-2d, 3d, 7a, and 7c, the torsion angles for the 16N, 17O substituents, which are important for a participation of the 17O substituent in the complexation of metal ions, have been determined. Furthermore, a preferred arrangement between the chelate ring and the steroid plane existed in all investigated condensation products attributable to torsion angles 16H-C16-16N-C of 5-61 degrees. This arrangement was also preserved in the copper(II) complex 11 with 16alpha,17beta-trans configuration of the bidentate steroid ligand and a ratio of 2:1 for ligand: copper in contrast with dimeric copper(II) complexes with a tridentate steroid ligand of 16beta, 17beta-cis configuration (ratio of 1:1 for ligand:copper). The crystal structures of the condensation products are also discussed. In most cases, intermolecular hydrogen bonds between 17-hydroxy groups and the chelate oxygen caused polymeric strands.

Aldehydes↗

A comparative study of the apical leakage of four root canal sealers and laterally condensed gutta-percha.

Previous studies have shown that lateral condensation of gutta-percha and sealer can provide an adequate apical seal. The purpose of this study was to compare the level of apical dye penetration when different sealers were used. One-hundred twenty-five teeth with single root canals were biomechanically prepared using the step-back technique and irrigation with EDTAC and 1% NaOCl solutions. The teeth were divided into five groups of 25 teeth each. The control group root canals were filled with laterally condensed gutta-percha and no sealer and the other four groups were filled with laterally condensed gutta-percha and either Apexit, Sealapex, Tubli-Seal, or AH26 sealer. After storage in 100% humidity at 37 degrees C for 48 h, the root surfaces were coated with nail varnish (except at the apex) and placed in 2% methylene blue dye solution and centrifuged at 3 x g for 3 min. The roots were sectioned transversely at 1-mm intervals to determine the following mean levels of dye penetrations: Apexit, 1.67 mm; Sealapex, 2.28 mm; Tubli-Seal, 1.95 mm; AH26, 0.82 mm; and gutta-percha alone, 8.37 mm. This study demonstrated that a root canal sealer should be used in conjunction with laterally condensed gutta-percha and that AH26 sealer provides a significantly better apical seal than the other sealers.

Bismuth↗

Humidification method that decreases condensate contamination in ventilator tubing.

OBJECTIVE: To demonstrate combining unheated bubble-through humidifier with a heat-moisture exchanger filter for the purpose of decreasing condensate contamination in mechanical ventilator tubing. DESIGN: Single-case, pretest and posttest. SETTING: University-affiliated and nonprofit hospital. PATIENT: A 32-year-old man with Pickwickian syndrome and pneumonia caused by Pseudomonas aeruginosa received mechanical ventilation for 14 days. METHODS: Ventilator tubing was cultured in two 24-hour trials, using a pretest and posttest design, to assess tubing bacterial contamination during use of 2 humidification methods. In the first trial, a traditional heated bubble-through humidifier (HBH) was used for 24 hours. Before the start of the second trial, the "wet" tubing and the heated bubble-through humidifier were removed and replaced with clean equipment through the use of aseptic technique. The bubble-through humidifier was placed on the "cold," or unheated mode, and a heat-moisture exchanger filter was attached to the Y-connector of the ventilator tubing. RESULTS: The heated bubble-through humidifier method revealed contamination of the ventilator tubing in 3 places with the patient's strain of P aeruginosa in addition to copious water condensate. The unheated bubble-through humidifier/heat moisture exchanger filter method demonstrated no bacterial contamination or condensate in the tubing. CONCLUSION: It can be inferred that the humidification method using the combination of an unheated bubble-through humidifier and a heat moisture exchanger filter has the potential benefit of preventing "reseeding" of the patient's airway with contaminated condensate.

Adult↗

Track formation and electron emission in swift ion collisions with condensed matter.

Electron ejection from target atoms is one of the basic processes when ionizing radiation interacts with matter. The primary electrons and their subsequent secondary interactions lead to the deposition of energy around the ion trajectory. The detailed knowledge of the structure of these ion tracks is a key issue for our understanding of radiation effects in condensed matter, an important example being calculations of the RBE of heavy particles, where doubly differential electron ejection cross sections are a key input parameter. Usually, data obtained from single collisions, i.e. with gas targets, are used, but condensed matter effects may considerably alter the emission patterns. Therefore, we will focus on the primary ionization (binary encounter and soft electron emission), which is common to single atoms (gas targets) and condensed matter. Then, special emphasis will be given to discuss effects, which are only seen in condensed matter (electron transport and multiple scattering effects, jet-like structures, wake effects due to collective excitation of plasmons), but not in gaseous targets (single collisions).

Electrons↗

Short-term changes in eating patterns explain the effects of condensed tannins on feed intake in heifers.

Ingestion of condensed tannins decreases feed intake in ruminants. Polyethylene glycol (PEG) forms high-affinity complexes with tannins. In two experiments carried out on Holstein heifers, quebracho (Q) from the Aspidosperma quebracho served as source of condensed tannins. The aims of the study were (i) to quantify the effect of Q on feed intake and eating behaviour in cattle fed complete mixed diets (CMDs); (ii) to clarify if changes induced in ingestive behaviour and feed intake by Q in cattle can be reversed by feeding PEG; and (iii) to clarify if the decrease in feed intake is associated with short-term (astringency, post-ingestive malaise) or longer-term effects. In experiment 1, 500 g/day of Q was found to be the minimal dose that decreased feed intake in heifers. A ratio of PEG:Q equal to 1:12.5 did not fully restore feed intake. In experiment 2, four heifers received a random sequence of four rations in a Latin-square design with feeding cycles of ca. 7 days: CMD containing no supplements (C), or supplemented with 625 g/day of Q without PEG (Q), with 625 g/day of Q and 250 g/day of PEG (Q-PEG), or with 250 g/day of PEG without Q (PEG). Individual rations were continuously weighed in the trough and the behaviour of heifers was observed for 180 min after distribution of CMD. Overall, feeding Q was associated with lowered feed intake and shorter duration of eating bouts, mainly of the first eating bout, immediately after distribution of the diet. A larger portion of the diet was consumed subsequent to 180 min after distribution in Q-fed heifers. Eating rate and the water to food ratio were not affected by Q. The effects of Q on feed intake were attenuated by feeding PEG. Heifers adapted effectively to condensed tannins by increasing the number of eating bouts and the portion of diet consumed subsequent to 180 min after distribution, so that no differences in feed intake were noted on the last day of each feeding cycle. Data are interpreted to show that: (i) negative effects of Q on feed intake derive from astringency of CT and short-term post-ingestive malaise; (ii) the increased number of eating bouts and their wider partition throughout the day are means to preserve the ruminal environment in Q-fed heifers; (iii) PEG has the potential to neutralize negative effects of condensed tannins in cattle.

Journal Article↗

The relative toxicity of compounds in mainstream cigarette smoke condensate.

Many different in vivo and in vitro tests are currently used to assess the toxicity of chemicals and complex mixtures such as cigarette smoke condensate. In vivo tests include assays in rodents to determine carcinogenicity, tumorigenicity and reproductive effects In vitro tests of mutagenicity are conducted with both bacterial and mammalian cell systems. A first step towards lowering the toxicity of cigarette smoke condensate is the identification of the relevant compound However, changing the concentration of a given smoke component may not linearly alter the biological activity of the complex mixture due to interactive effects. The "effective toxicity" of a chemical constituent is a function of the concentration, the metabolic fate, the potency in in vivo and in vitro assays, and the ability to reach the target tissues. The logarithm of the octanol-water partition coefficient (log P) is an important parameter since it affects metabolism, biological transport properties and intrinsic toxicity. Using concentration data from the International Agency for Cancer Research (IARC), biological activity data from the Registry of Toxic Effects of Chemical Substances (RTECS) database and measured and calculated log P values, we have rank ordered some of the important compounds in cigarette smoke condensate by their measured or potential toxicity. Condensates from different cigarette brands, tar categories and styles vary in their concentrations of these compounds. Chemicals of greater commercial or scientific interest may be toxicity tested more extensively, thereby increasing the probability of positive test results and highlighting the need for consideration of structure-activity relationships.

Acetaldehyde↗

SMC proteins in bacteria: condensation motors for chromosome segregation?

SMC proteins are a ubiquitous protein family, present in almost all organisms so far analysed except for a few bacteria. They function in chromosome condensation, segregation, cohesion, and DNA recombination repair in eukaryotes, and can introduce positive writhe into DNA in vitro. SMC proteins and the structurally homologous MukB protein are unusual ATPases that form antiparallel dimers, with long coiled coil segments separating globular ends capable of binding DNA. Recently, SMC proteins have been shown to be essential for chromosome condensation, segregation and cell cycle progression in bacteria. Identification of a suppressor mutation for MukB in topoisomerase I in Escherichia coli suggests that SMC proteins are involved in negative DNA supercoiling in vivo, and by this means organize and compact chromosomes. A model is discussed in which bacterial SMC proteins act after an initial separation of replicated chromosome origins into the future daughter cell, separating sister chromatids by condensing replicated DNA strands within both cell halves. This would be analogous to a pulling of DNA strands into opposite cell halves by a condensation mechanism exerted at two specialised subregions in the cell.

Bacillus subtilis↗

The application of fluorescence correlation spectroscopy in detecting DNA condensation.

We report the application of fluorescence correlation spectroscopy (FCS) in characterizing conformational changes (condensation) of chemically well-defined DNA plasmids. The plasmids: pHbetaAPr-1-neo (10 kbp, contour length 3.4 microm) and pBluescript SKt (2.96 kbp, contour length 1.02 microm) were imaged by a confocal fluorescence microscope using two fluorescent probes: ethidium bromide (EtBr) and propidium iodide (PrIo). It became clear that the DNA molecule exhibits discrete conformational change between the coil and globule states with the addition of a small amount (the order of magnitude being 10(-5) M) of cationic surfactant, spermine and hexadecyltrimethyl ammonium bromide (HTAB). When the concentrations of both condensing agents are smaller than 6.0x10(-6) M and 2.0 x 10(-6) M for the 10 and 2.96 kbp, both plasmids are in the extended coil state with diffusion constants D(10 kbp)=9.6 x 0(-13) m(2) s(-1) and D(2.96 kbp)=2.5x10(-12) m(2) s(-1), respectively. When the condensing agent in a concentration higher than 1.10 x 10(-5) M is added to pHbetaAPr-1-neo (10 kbp), plasmids are in the condensed globular state and their diffusion constants are D(10 kbp)=8.0 x 10(-12) m(2) s(-1) (spermine) and D(10 kbp)=5.5x10(-12) m(2) s(-1) (HTAB). The globular state of the pBluescript SKt (2.96 kbp) plasmids is characterized by diffusion constants equal to D(2.96 kbp)=9.2x10(-12) m(2) s(-1) (spermine) and D(2.96 kbp)=8.2x10(-12) m(2) s(-1) (HTAB).

Algorithms↗

Electrostatic free energy of the DNA double helix in counterion condensation theory.

Polyelectrolyte theory based on counterion condensation is extended from the standard line charge model to helical and double helical charge arrays. The number of condensed counterions turns out to be the same as for a line charge with charge density equal to the axial charge density of the helix. Also, the logarithmic salt dependence of the electrostatic free energy is the same in the range of lower salt concentration, so that the limiting laws remain unchanged. However, the internal free energy of the condensed layer of counterions and the overall electrostatic free energy depend on the helical parameters. At higher salt, the free energies of both single and double helix are negative, indicating electrostatic stabilization of the helical charge lattices due to the mixing entropy of the condensed counterions. Except at very low salt, the free energy of a single helix is higher than the free energy of a double helix with twice the charge density. With B-DNA parameters and single strands modeled as single helices, the predicted salt dependence of the free energy of transition from double helix to separated single strands has a maximum at approximately 0.2 M salt, close to the location in the laboratory of this well-known feature of the DNA strand separation transition. We also calculate the electrostatic free energy for the transition of the DNA double helix from the B to the A conformation. The B form is electrostatically stable over most of the salt range, but there is a spontaneous electrostatic transition to A near 1 M salt. The electrostatic free energy values are close to the experimental values of the overall (electrostatic plus non-electrostatic) transition free energies for A-philic base pair sequences. We are led to suggest that the experimentally observed B-to-A transition for A-philic sequences near 1 M salt in water is governed by the polyelectrolyte properties of these two conformations of the DNA double helix. The effect of ethanol, however, cannot be attributed to lowering of the bulk dielectric constant.

DNA↗