[Fascial closure of the abdominal wall by dynamic suture after topical negative pressure laparostomy treatment of severe peritonitis--results of a prospective randomized study]. / Uzáver steny brisní po laparostomii s pouzitím negativníiho tlaku pro tezkou peritonitidu pomocí dynamické fasciální sutury--výsledky prospektivní randomizované studie.

INTRODUCTION: Severe peritonitis is a frequent condition characterized by high morbidity and mortality rates. Topical negative pressure (TNP) laparostomy could improve the results of the treatment, provided that the adverse events of this method are reduced. The aim of our study was to prove, in a prospective randomized study, that the primary use of TNP laparostomy reduces morbidity and mortality when compared to primary abdominal wall closure after the index surgery for severe peritonitis. The possibility of the abdominal wall fascial closure significantly influencing morbidity was the main topic of this study. MATERIAL AND METHODS: Between 9/2009 and 9/2011,57 patients with severe peritonitis were included in the study at the Department of Surgery of the Bulovka Faculty Hospital; 28 of them were randomized to the TNP laparostomy group and 29 to the primary closure group. The two groups did not differ in age, gender, polymorbidity and severity of peritonitis. RESULTS: The length of hospital stay was similar in both groups (median: 22 days; range 10-171 days) in the intervention group and 23 days (range 3-71) in the control group (p = 0.89). The mortality rate was significantly lower in the TNP laparostomy group in comparison with the primary closure group (3 patients, 11% vs. 12 patients, 41%; p = 0.01). A complete closure of the abdominal wall including fascia and complete abdominal wall healing was achieved in 80% of survivors in the TNP group, compared to 29% in the primary closure group (p = 0.01). No enteral fistula occurred in any surviving patients from both groups. The overall length of abdominal wall healing was significantly shorter in the TNP group (median: 7; 7-94 days, versus 30; 7-223; p = 0.04). CONCLUSIONS: Primary TNP laparostomy is an effective and safe method in the treatment of severe peritonitis. Keeping good clinical practice, especially using dynamic suture as early as after the index surgery and the timely closure of laparostomy as soon as the indication disappears (according to relevant criteria) leads to a significantly higher abdominal wall healing rate, icluding fascial closure, than after peritonitis treatment without laparostomy.

Molecular identification and phylogenetic relationship of flying fishes of Tamil Nadu coast for fishery management purposes.

The partial sequences of cytochrome c oxidase subunit I (COI) mitochondrial gene were analysed for developing species specific molecular signatures and phylogenetic relationship among the commercially important flying fishes (Cheilopogon cyanopterus, Cheilopogon furcatus and Hirundichthys coromandelensis) distributed in Tamil Nadu coast. Accurate identification of these species is important for fishery management as its morphological characters are very similar. Since the morphological features are very similar, accurate identification using molecular tools is essential for sustainable utilization and management of these species across their distributional range. The estimated transition/transversion bias (R) is 3.45. The average nucleotide sequences calculated were A = 30.00%, T/U = 26.40%, C = 17.00% and G = 26.60%. Using COI data analysis, the intraspecies genetic distance ranged from 0.00 to 0.05, while it varied from 0.06 to 0.08 for interspecies. Partial sequences of the genes provided sufficient phylogenetic information to distinguish the three flying fishes indicating the usefulness of mtDNA-based approach in species identification.

Graves' disease and radioiodine therapy. Is success of ablation dependent on the achieved dose above 200 Gy?

AIM: This study was performed to determine the results of ablative radioiodine therapy (RIT) when the achieved dose in the thyroid was above 200 Gy and to characterize predictive factors for treatment outcome. PATIENTS, METHODS: A total of 571 consecutive patients were observed for 12 months between July 2001 and June 2004. Inclusion criteria were a confirmed diagnosis Graves' disease, compensation of hyperthyroidism and withdrawal of antithyroid drugs two days before preliminary radioiodine-testing and RIT. The intended dose was 250 Gy and the therapeutically achieved dose was calculated from serial uptake measurements. The end-point measure was thyroid function 12 months after RIT; success was defined as elimination of hyperthyroidism. The relation between success rate and the achieved dose, thyroid volume, age and sex of patients, TSH- and TRAb-values and presence of ophthalmopathy was analysed. RESULTS: Relief from hyperthyroidism was achieved in 96% of patients who received more than 200 Gy, even for thyroid volumes >40 ml. The success of ablative RIT was not influenced by age or sex of patients, or by TSH- or TRAb values or concomitant ophthalmopathy. The mean achieved dose in the thyroid was 298 Gy with a standard deviation of 74.6 Gy. CONCLUSION: To achieve a dose of over 200 Gy with the above standard deviation, we recommend calculating an intended dose of 250 Gy and using a dosimetric approach with early and late uptake values in the radioiodine test, to allow early therapeutic intervention should the posttherapeutic thyroid dose fall unexpectedly below 200 Gy.

Experimental and theoretical study of the neutron dose produced by carbon ion therapy beams.

High-energy (12)C ions offer favourable conditions for the treatment of deep-seated local tumours. Several facilities for the heavy ion therapy are planned or under construction, for example the new clinical ion-therapy unit HIT at the Radiological University Clinics in Heidelberg. In order to improve existing treatment planning models, it is essential to evaluate the secondary fragment production and to include these contributions to the therapy dose with higher accuracy. Secondary neutrons are most abundantly produced in the reactions between (12)C beams and tissues. The dose contribution to tissues by a neutron is fairly small compared with the projectile and the other charged fragments due to no ionisation and the small reaction cross-sections; however, it distributes in a considerably wider region beyond the bragg-peak because of the strong penetrability. Systematic data on energy spectra and doses of secondary neutrons produced by (12)C beams using water targets of different thicknesses for various detection angles have therefore been measured in this study at GSI Darmstadt.

Non-satellite repetitive human DNA families. Sequence properties and evidence for occurrence in chimpanzee DNA.

Repetitive human DNA, fractionated on CsCl gradients following hydroxyapatite isolation, contains two complex DNA fractions, the 1.703 and 1.714 DNA families (Marx, K.A., Allen, J.R. and Hearst, J.E. (1976) Biochim. Biophys. Acta 425, 129-147). Biphasic Topt profiles, obtained in DNA excess hybridizations with cRNA tracers from each DNA family, have been shown to be the likely result of a fast kinetic component hybridizing at higher temperatures (67 degrees C peak) and this fast plus a slow kinetic component both hybridizing at lower temperatures (47 degrees C peak). Equilibrium CsCl gradient DNA-cRNA hybrid distributions support previous interpretations of the sequence composition of both DNA families. That is, the fast component is a relatively undiverged repetitive sequence of recent origin, while the slow component is a highly diverged, less thermally stabile, old primate sequence. This interpretation is further strengthened by cRNA tracer hybridization experiments in chimpanzee DNA excess where the fast component appears to be absent and the slow component present.

Ion competition and micrococcal nuclease digestion studies of spermidine-condensed calf thymus DNA. Evidence for torus organization by circumferential DNA wrapping.

Spermidine-condensed calf thymus DNA structures have been studied by ion competition using a sedimentation assay and by micrococcal nuclease digestion. Competitor ions Mg2+, Ca2+ and putrescine2+ show specific ion effects; but all three appear to affect the DNA condensation-decondensation equilibrium caused by spermidine3+ in a qualitatively similar manner, suggesting the spermidine3+-DNA interaction is largely electrostatic. Our data show a hysteresis in condensation and decondensation transition directions. We interpret this in terms of a kinetic block in the condensation direction with decondensation representing the equilibrium state of the system. These results agree with results obtained from related systems using different measurement techniques. Micrococcal nuclease digestion of spermidine-condensed calf thymus DNA produces broad but discrete bands in gel electrophoresis experiments. At least two bands determined to be 760 +/- 87 bp and 1355 +/- 135 bp, possess the size ratio 1:1.8 +/- 0.4 consistent with their forming the monomer and dimer fragments of an arithmetic band series. We rationalize this result in terms of a localized micrococcal nuclease cleavage model of circumferentially-wrapped DNA toruses proposed previously by Marx, K.A. and Reynolds, T.C. (Proc. Natl. Acad. Sci. (1982) 79, 6484-6488). The arithmetic series monomer band (760 +/- 87 bp), corresponding to wrapping B DNA once circumferentially about the torus, is in agreement with the electron microscopic measurements of hydrated calf thymus DNA torus circumferences presented by Marx, K.A. and Ruben, G.C. (Nucleic Acids Res. (1983) 11, 1839-1853).

High-affinity microtubule protein-higher organism DNA complexes. Many-fold enrichment in repetitive mouse DNA sequences comprised of satellite DNAs.

We have examined aspects of the interaction of cycled microtubule protein preparations with 35S-labeled mouse DNA tracer in a competition system with unlabelled competitor E. coli or mouse DNA. The nitrocellulose filter binding assay was used to measure interaction by scintillation counting. DNA molecular weight affected the levels of filter retained 35S-labelled mouse tracer DNA. Filter retention levels increased if 35S-labelled mouse DNA tracer size was increased, and the filter binding level decreased if competitor DNA size was increased. There was a sizeable, reproducible difference in the 35S-labelled mouse DNA tracer binding level of about 1% when E. coli or mouse DNA competitors were compared. Mouse DNA more effectively competed with 35S-labelled mouse DNA for microtubule protein binding than did E. coli DNA, suggesting that a small class of higher-organism DNA sequences interacts very strongly with microtubule protein. From other studies we know this to be the MAP fraction (Marx, K.A. and Denial, T. (1984) in The Molecular Basis of Cancer (Rein, R., ed.), Alan R. Liss, New York, in the press; and Villasante, E., Corces, V.G., Manso-Martinez, R. and Avila, J. (1981) Nucleic Acids Res. 9, 895-908). We find that this difference in competitor DNA strength is qualitatively similar under high-stringency conditions (0.5 M NaCl, high competitor [DNA]) we developed for examining high-affinity complexes. Under high-stringency conditions we isolated 1.2% and 0.6% of 35S-labelled mouse DNA at 4200 and 350 bp respective sizes as nitrocellulose filter bound DNA-protein complexes. At both molecular weights these high-affinity DNA sequences, isolated from the filters, were shown to be significantly enriched in repetitive DNA sequences by S1 nuclease solution reassociation kinetics. The kinetics are consistent with about a 4-fold mouse satellite DNA enrichment as well as enrichment in other repetitious DNA sequence classes. The high molecular weight filter-bound DNA samples were sedimented to equilibrium in CsCl buoyant density gradients and found to contain primarily mouse satellite DNA density sequences (1.691 g/cm3) with some minor fractions at other density positions (1.670, 1.682, 1.705, 1.740, 1.760 g/cm3) similar to those observed by our laboratory in previous investigations of micrococcal nuclease-resistant chromatin (Marx, K.A. (1977) Biochem. Biophys. Res. Commun. 78, 777-784). That the high-affinity microtubule-bound DNA was some 3-5-fold enriched in mouse satellite sequences was demonstrated by its characteristic BstNI restriction enzyme cleavage pattern.

A novel metabolic pathway for leukotriene B4 in different cell types: primary reduction of a double bond.

Addition of leukotriene B4 together with trace amounts of tritiated leukotriene B4 to different cell types, such as bone marrow-derived macrophages, T-lymphocytes, mesangial cells or fibroblast tumor cells, led to the formation of several hitherto unknown degradation products within hours. None of them could be identified as 20-hydroxy- or 20-carboxyleukotriene B4, known to be produced by polymorphonuclear leukocytes. The primarily formed transient leukotriene B4 metabolite was less polar than leukotriene B4 and was detectable by measuring its ultraviolet absorbance at 232 nm or its radioactivity. Mass spectral analysis showed very similar fragmentation spectra of leukotriene B4 and its primary metabolite. The most abundant ion and the main fragments of the new metabolite were increased by two mass units compared to leukotriene B4. These observations suggest that, in a variety of cells, leukotriene B4 is first reduced to a 5,12-dihydroxyeicosatrienoic acid, which is further converted to secondary hydrophilic degradation products. This raises the question of the major route of leukotriene B4 metabolism in vivo.

Characterization of the repetitious human DNA families.

Human DNA isolated from HeLa cells or human placental tissue has been fractionated on hydroxyapatite at COt 1.0. The 25% of total DNA isolated at COt 1.0 is composed of 3% foldback DNA and 22% which renatures by second-order kinetics and can be resolved into five renatured DNA families banding at distinct densities in CsC1 gradients. The individual renatured DNA families were isolated and their physical properties including reassociation kinetics determined. A two-component kinetic analysis was used to resolve kinetic heterogeneity. The three lightest density DNA families possess satellite DNA-like properties. The two heaviest density DNA families were shown to contain reassociated highly repetitious DNA as well as single-stranded, middle-repetitious DNA sequences, suggesting interspersion. The middle repetitious DNA sequences are thought to be related in these two DNA families.

Spinal cord contusion in the rat: somatosensory evoked potentials as a function of graded injury.

A weight-drop technique was used to produce mild, moderate, or severe spinal cord contusive injury in rats. At 4 weeks after injury, somatosensory evoked potentials (SEPs) were recorded with silver ball electrodes placed over the somatosensory cortex of anesthetized rats to measure the response to sciatic nerve stimulation. Both SEP area and amplitude were measured and were highly correlated with each other. Both indices of the SEP correlated inversely with the height of the weight drop and directly with the degree of residual function assessed at 4 weeks after injury. Measures of residual function consisted of a motor score, inclined plane test, and a combined behavioral score based on several neurologic functions. No correlation between latency of the SEP with degrees of contusive injury was observed. The data indicate that the SEP can be used as one criterion in the assessment of the severity of a lesion in a rat model of a graded spinal cord injury.

DNA bound to polypyrrole films: high-resolution imaging, DNA binding kinetics and internal migration.

We have studied the time-dependent uptake of 35S radiolabeled DNA with electrochemically prepared polypyrrole films. The two distinct polypyrrole film surfaces, a rough (solution polymeric growth face, R) and a smooth surface (electrode face, S) were characterized by low-resolution AFM and high-resolution transmission electron microscopy (TEM). These studies showed the presence of steep contours and defects in the form of large and small surface holes and valleys on the rough surface of polypyrrole. The void dimensions ranged from the nanoscale to micron size. By contrast, the smooth surface was flatter and largely devoid of significant structural defects and exhibited closer packing of the polypyrrole chains over large areas. Both surfaces were comprised largely of chains whose average diameters were 1.0-1.2 +/- 0.3 nm. The surface characterization studies were complemented by time-dependent DNA uptake studies which showed a t1/2-dependent total uptake of 35S DNA at higher levels on the rough surface compared to the smooth surface. This is consistent with the apparent higher effective surface area of the rough surface compared to the smooth. Using a proportional counter the time-dependent ratio (R/S) of the 35S DNA detected from the rough surface of the polypyrrole disk to that detected from the smooth surface suggested that DNA was migrating into the disk interior from its uptake surface. The rough side defect dimensions measured by TEM were more than sufficient to allow for the penetration and migration of DNA into the disk interior. Both R/S ratios were extrapolated and found to intersect at an R/S value close to 1.0, suggesting a kinetic process leading ultimately towards a nearly uniform radiolabeled DNA distribution in the disk. These kinetic results were in agreement with the surface characterization studies and suggest a model in which sizeable internal pores exist throughout the electrochemically prepared polypyrrole, that could account for the DNA migration effect. This was confirmed by TEM of the interior of a polypyrrole disk produced by Argon ion milling.

8-phenyl-10,10a-dihydropyrido

Base treatment of the pyridinium bromides 11a-e gives rise to the formation of the dihydropyridoazepines 14a-e as the only monomolecular products. The reaction takes place by initial deprotonation to the ylides 12, which undergo 8pi-electrocyclization affording the seven-membered-ring systems; no products of a dipolar 6pi-cyclization were detected. On the basis of quantum mechanical calculations a rationalization of the periselectivity of the electrocyclization process is given.

Human monocytes convert leukotriene B4 to two dihydro-leukotriene B4-metabolites.

Human monocytes metabolize LTB4 by an additional pathway different from omega-oxidation. Reverse-phase high performance liquid chromatography showed four metabolites: 20-COOH-LTB4, 20-OH-LTB4 and two metabolites less polar than LTB4 with an UV maximum at 232 nm. Gas-chromatography mass-spectrometry showed nearly identical mass spectra for both metabolites. The main mass fragments of the two metabolites were increased by two mass units compared to LTB4. Our findings suggest that LTB4 had been reduced to a known and a new dihydro-metabolite of LTB4. Both metabolites together amounted to 85% of total metabolites. The remaining 15% were omega-oxidation products. Thus, the major pathway of LTB4 metabolism by human monocytes is reduction to dihydro-LTB4.

A quartz crystal microbalance cell biosensor: detection of microtubule alterations in living cells at nM nocodazole concentrations.

The quartz crystal microbalance (QCM) was used to create a piezoelectric biosensor utilizing living endothelial cells (ECs) as the biological signal transduction element. ECs adhere to the hydrophilically treated gold QCM surface under growth media containing serum. At 24 h following cell addition, calibration curves were constructed relating the steady state Deltaf and DeltaR shift values observed to the numbers of electronically counted cells requiring trypsinization to be removed from the surface. We then utilized this EC QCM biosensor for the detection of the effect of [nocodazole] on the steady state Deltaf and DeltaR shift values. Nocodazole, a known microtubule binding drug, alters the cytoskeletal properties of living cells. At the doses used in these studies (0.11-15 microM), nocodazole, in a dose dependent fashion, causes the depolymerization of microtubules in living cells. This leads a monolayer of well spread ECs to gradually occupy a smaller area, lose cell to cell contact, exhibit actin stress fibers at the cell periphery and acquire a rounded cell shape. We observed the negative Deltaf shift values and the positive DeltaR shift values to increase significantly in magnitude over a 4-h incubation period following nocodazole addition, in a dose dependent fashion, with a transition midpoint of 900 nM. Fluorescence microscopy of the ECs, fixed on the gold QCM surface and stained for actin, demonstrated that the shape and cytoskeleton of ECs were affected by as little as 330 nM nocodazole. These results indicate that the EC QCM biosensor can be used for the study of EC attachment and to detect EC cytoskeletal alterations. We suggest the potential of this cellular biosensor for the real time identification or screening of all classes of biologically active drugs or biological macromolecules that affect cellular attachment, regardless of their molecular mechanism of action.

Chromosomal aberrations in couples undergoing intracytoplasmic sperm injection: influence on implantation and ongoing pregnancy rates.

OBJECTIVE: To determine the incidence of chromosomal aberrations in couples undergoing intracytoplasmic sperm injection (ICSI) and their influence on subsequent implantation and ongoing pregnancy rates. DESIGN: Prospective study. SETTING: Fertility center. PATIENT(S): Candidates for ICSI. INTERVENTION(S): Chromosomes were trypsin-banded in 2,280 patients. In all cases, 10 metaphases were karyotyped. Sex chromosome analysis was performed in 10 additional metaphases. When apparent chromosomal aberrations were detected, 100 metaphases were analyzed. MAIN OUTCOME MEASURE(S): Implantation and ongoing pregnancy rates in couples with a chromosomal disorder. RESULTS: A chromosomal abnormality was demonstrated in 7.2% of all couples. Among the male partners, 4.48% had aberrations. Autosomal aberrations were present in 2.96%, and numerical or structural sex chromosome abnormalities were found in 1.52%. Among the female partners, numerical or structural abnormalities were documented in 9.79%. Only 2.32% of the female partners had autosomal structural abnormalities. Numerical or structural anomalies involving sex chromosomes were found in 7.47%. Implantation rates of 9.4% and 16.3% per embryo were observed in female partners with sex chromosome mosaicism and autosomal aberrations, respectively. In male partners, the respective rates were 3.8% and 23.1%. CONCLUSION(S): The incidence of chromosomal disorders in couples seeking ICSI treatment is considerable, especially minor mosaicism (<10%) of sex chromosomes in the female partners. Preliminary data indicate a low implantation rate in couples with minor mosaicism of sex chromosomes.

Involvement of the Na,K-ATPase in the induction of ion channels by palytoxin.

The effects of ouabain, ATP, and vanadate on palytoxin induction of ion channels were examined with the aim of elucidating the role of Na,K-ATPase in palytoxin action. Palytoxin-induced membrane depolarization of crayfish giant axons and single channel currents of frog erythrocytes and mouse neuroblastoma N1E-115 cells were examined using the intracellular microelectrode and patch-clamp techniques. External application of palytoxin in nanomolar concentrations induced depolarization in the crayfish giant axons, and the depolarization was inhibited by pretreatment of the axon with ouabain (10 microM). Internally perfused axons were less sensitive to palytoxin unless ATP (6 mM) was added internally. In patch-clamp experiments, picomolar palytoxin in the patch electrode induced single channels in both cell-attached and inside-out patches of erythrocytes and neuroblastoma cells. The induced channels had a conductance of about 10 pS, reversed near 0 mV in physiological saline solution, and was permeable to Na+, K+, Cs+, and NH4+, but not to choline. Single channel activities induced by palytoxin were inhibited by ouabain (10 microM) and vanadate (1 mM), but promoted by ATP (1 mM). The modulating effects of ouabain, vanadate, and ATP on palytoxin action suggest that the Na,K-ATPase is involved in the induction of single channels by palytoxin. Palytoxin-induced and ouabain-inhibitable single channels were observed in planar lipid bilayer incorporated with purified Na,K-ATPase. The results indicate that an interaction between palytoxin and Na,K-ATPase leads to opening of a 10-pS ion channel. They further raise the possibility that a channel structure may exist in the sodium pump which is uncovered by the action of palytoxin.

A study of phi X-174 DNA torus and lambda DNA torus tertiary structure and the implications for DNA self-assembly.

Hydrated torus shaped complexes were examined by transmission electron microscopy in both spermidine-condensed linear and nicked circular phi X-174 DNA and lambda DNA preparations. Freeze-etch replicas of both these torus samples, produced with very low Pt metal deposition levels (9APt/C), were found to have circumferentially wound single DNA double helix size surface fibers in the range of 30A width. Measurements of torus inner and outer circumference as well as ring thickness were performed. Observed differences in the torus dimension distributions from circular phi X-174 DNA and linear phi X-174 DNA may be related to the different topological constraints on DNA folding in these two samples (1). On the basis of annulus thickness measurements phi X-174 DNA toruses, in contrast to lambda DNA toruses, were observed to fall into two classes identified as being formed from monomer DNA condensation and multimer DNA condensation. All of the torus substructure and population dimensions observed here are consistent with the continuous circumferential DNA winding model of torus organization proposed by Marx and Reynolds (1) to explain the micrococcal nuclease cleavage properties of the toruses. End-on view measurements of the torus thickness were made from micrographs obtained by extensive tilting of the object replica. These direct measurements confirmed quaternary structure interpretations made from simple strand packing models. We compared the measured torus properties in this linear DNA size series (5386-48000 bp). With increasing DNA length the pattern of DNA strand self-assembly was found to be more varied producing lambda DNA toruses of varying shape. The relevance of our study to the problem of lambda bacteriophage DNA head packaging was discussed.

The affinity of DNA-microtubule protein complexes and their disruption by tubulin binding drugs.

Using the gel shift assay system, we have measured the apparent affinity constant for the interaction of two different DNAs with MAP proteins found in both total calf brain microtubules and heat stable brain preparations. Both DNAs studied contained centromere/kinetochore sequences- one was enriched in the calf satellite DNA; the other was a large restriction fragment containing the yeast CEN11 DNA sequence. Complexes formed using both DNAs had similar Kapp values in the range of 2.1 x 10(7) M-1 to 2.0 x 10(8) M-1. CEN11 DNA-MTP complexes had by far the highest Kapp value of 2.0 x 10(8) M-1. The CEN11 DNA sequence is where the yeast kinetochore of chromosome 11 is formed and where the single yeast microtubule is bound in vivo. The CEN11 conserved region II known binding sites-(dA/dT)n runs- for mammalian MAP2 protein, are in good agreement with this higher Kapp value. The effects of the classical tubulin binding drugs colchicine, podophyllotoxin and vinblastine on the DNA-MAP protein complex stability were investigated by determining the drug concentrations where the complexes were destabilized. Only the complexes formed from total microtubule protein (tubulin containing) were destabilized over a wide drug concentration range. Heat stable brain protein complexes (no tubulin) were largely unaffected. Furthermore, it took 10-100 fold higher drug concentrations to disrupt the CEN11 DNA complexes compared to the calf thymus satellite DNA enriched complexes. These data support our previous results suggesting that there is a DNA sequence dependent interaction with MAP proteins that appears to be conserved in evolution (Marx et. al., Biochim. Biophys. Acta. 783, 383-392, 1984; Marx and Denial, Molecular Basis of Cancer 172B, 65-75 1985). In addition, these results imply that the classical tubulin binding drugs may exert their biological effects in cells at least in part by disrupting DNA-Protein complexes of the type we have studied here.

Studies of DNA organization in hydrated spermidine-condensed DNA toruses and spermidine-DNA fibres.

We have investigated in vitro spermidine-condensed DNA preparations by both biochemical and freeze-etch electron microscopic approaches. These studies lead us to the conclusion that the reversibly condensed preparations, qualitatively described by Manning's counterion condensation theory, contain disk-like torus structures largely comprised of unidirectional, circumferentially wrapped DNA. Stereoscopic measurements on stereomicrographs of DNA torus and fibre objects have demonstrated the feasibility of directly measuring DNA writhe or, for that matter, mapping any secondary, tertiary or quaternary structure features of a hydrated macromolecular array in which the features can be differentially highlighted by low replica metal shadow levels.

Alignment of (dA).(dT) homopolymer tracts in gene flanking sequences suggests nucleosomal periodicity in D. discoideum DNA.

It has been shown that the frequency versus size distribution of A and T overlapping and non-overlapping homopolymer tracts of N > 5 in D. discoideum gene flanking and intron regions are significantly greater than in coding regions(1). In the present report, we demonstrate, that a spatial periodicity exists in long A and T tracts (N > 10) in long flanking sequences by scored alignments of those tracts (N > 10) with the nucleosomal repeat. A tract spacing was found at 185-190 bp that corresponds to a maximum alignment score. This is exactly the average spacing of D. discoideum nucleosomes determined experimentally. A majority of A and T tracts in flanking sequences are often spaced by short DNA stretches and the total length of adjacent A and T tracts plus the interrupting short DNA stretch corresponds closely to the average experimentally measured nucleosomal linker DNA size in D. discoideum-42 bp. These data suggest a model which has A and T runs of N > 10 bp in flanking DNA of D. discoideum organized in a regular phase with nonhomopolymer sequences along the DNA. This model has functional implications for A and T tracts, suggesting that they are found in nucleosomal linker DNA regions of chromatin during some necessary portion(s) of the life of the cell.