Effects of Na+ and Mg2+ on the structures of supercoiled DNAs: comparison of simulations with experiments

Recent cryo-electron microscopy (cryo-EM) results suggest that sufficient NaCl concentration (> or approximately 0.1 M) and superhelix density (> or approximately-0.05) cause circular DNAs to adopt highly extended, tightly interwound configurations, in which the strands are laterally contiguous along almost their entire length. Millimolar levels of MgCl2 reportedly act synergistically with NaCl to produce similar conformations. However, Monte Carlo simulations with purely repulsive interduplex forces failed to reproduce such structures. In the present work, solution measurements of particular physical properties were performed both to characterize the effects of Na+ and Mg2+ on DNA structure and to provide quantitative tests of Monte Carlo simulations of circular DNAs. Supercoiled p30 delta DNAs in 10 mM Tris plus 0, 0.122, and 0.1 M NaCl, and 0.1 M NaCl plus 4 mM Mg2+ were examined by static and dynamic light scattering (LS and DLS), time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium, and circular dichroism (CD) spectroscopy. Upon addition of 0.122 M NaCl, the radius of gyration (Rg) decreased substantially, which indicates that p30 delta adopts a more compact structure. This contradicts the cryo-EM studies, where molecular extension and Rg both increase upon adding 0.1 M NaCl. In 0.1 M NaCl, the torsion constant measured by FPA is practically invariant to superhelix density, and the plateau diffusion coefficient at large scattering vector (Dplat) is likewise nearly the same at both relaxed and native superhelix densities. Such invariance is difficult to reconcile with any transition from relaxed circles to tightly interwound structures with laterally contiguous strands. Metropolis Monte Carlo simulations were performed to generate canonically distributed sets of structures, from which average Do values and scattered intensity ratios, [symbol: see text]I (zero) [symbol: see text]/[symbol: see text] l(k) [symbol: see text], were calculated. Agreement between simulations and experiments in regard to [symbol: see text] I(O) [symbol: see text] /[symbol: see text] I(k) [symbol: see text], D(zero) and the supercoiling free energy, delta Gsc (delta l), is remarkably good for the most extensively studied p30 delta samples. The simulated structures exhibit no sign of very tight interwinding with extensive lateral contacts, but instead exhibit most probable superhelix diameters of 85 to 90 A. When 4 mM Mg2+ was added to native supercoiled p30 delta in 0.1 M NaCl, Rg decreased, D(zero) increased, and the longest internal relaxation rate (1/tau 2(zero)) increased, all of which indicate a further overall contraction of the molecular envelope. The torsion constant exhibited a slight increase that is hardly statistically significant. In this case, agreement between the simulations and experiments was only semi-quantitative for most samples investigated, although the predicted contraction was exhibited by all five samples of p30 delta and one of pBR322 DNA. The simulated structures in 0.1 M NaCl plus 4 mM Mg2+ again showed no sign of extensive lateral contacts. A plausible explanation is proposed for the highly extended, tightly interwound structures seen in cryo-EM, and explicitly tested by Monte Carlo simulations of a 1000 bp circular DNA at +25 and -50 degrees C. Structures identical to those seen in cryo-EM are in fact the equilibrium structures in the simulations at -50 degrees C, and the estimated time for equilibration (2.3 x 10(-6) second) is much smaller than the estimated time for vitrification (1 x 10(-4) second).     

A phase II trial of amonafide in patients with nonsquamous cell carcinoma of the cervix. A Gynecologic Oncology Group study

Twenty-seven patients with nonsquamous cell carcinoma of the cervix were entered into a Phase II study of amonatide; 24 patients were evaluable for toxicity, while 23 were evaluable for response. Patients received amonafide, 300 mg/m2, intravenously for 5 consecutive days every 3 weeks. The median age of patients was 45 years. All but two patients were completely ambulatory. Twelve patients had received prior chemotherapy, while 22 had been treated with radiation therapy. One of 27 (4.3%) patients had a partial response (PR) to this regimen and 13 (56.5%) had stable disease. Sixteen patients experienced a median white blood cell (WBC) nadir of 350/mm3, seven developed life-threatening thrombocytopenia, and one had severe anemia requiring transfusion. Nonhematologic toxicity was mild. Amonafide had insignificant activity in these patients with nonsquamous cell carcinoma of the cervix.     

Optimisation of antibiotic therapy in cystic fibrosis patients. Pharmacokinetic considerations

Antibiotic therapy plays a central role in the medical management of patients with cystic fibrosis. While totally convincing efficacy data are lacking, antibiotics probably have a pronounced beneficial effect on both morbidity and mortality. Much has been learned in the past 20 years about antibiotic use in this population. At the same time, new antimicrobial agents with the potential to treat this condition have become available for use. The pharmacokinetics of a number of antibiotic classes, including beta-lactams, aminoglycosides and quinolones, are altered in this patient population. Increased total body clearance is a common occurrence but is not always changed enough to warrant altered dosages. Nonetheless, in light of altered pharmacokinetics in the cystic fibrosis population, appropriate dosage and monitoring parameters for a number of antibiotics have been determined.     

RNA tertiary structure mediation by adenosine platforms

The crystal structure of a group I intron domain reveals an unexpected motif that mediates both intra- and intermolecular interactions. At three separate locations in the 160-nucleotide domain, adjacent adenosines in the sequence lie side-by-side and form a pseudo-base pair within a helix. This adenosine platform opens the minor groove for base stacking or base pairing with nucleotides from a noncontiguous RNA strand. The platform motif has a distinctive chemical modification signature that may enable its detection in other structured RNAs. The ability of this motif to facilitate higher order folding provides one explanation for the abundance of adenosine residues in internal loops of many RNAs.     

Interferon-gamma inhibits 35S incorporation in heparan sulfate synthesized by human skin fibroblasts

Glycosaminoglycans synthesized by human skin fibroblasts were simultaneously radiolabelled with D-[1-(3H)]glucosamine and Na2(35)SO4. Considering 3H incorporation, we found that IFNgamma increased the production of glycosaminoglycan synthesis, including hyaluronic acid, heparan and chondroitin/dermatan sulfate. In contrast, the production of heparan and chondroitin/dermatan sulfate was slightly decreased on the basis of the 35S signal. Furthermore, when heparan sulfate was treated with nitrous acid, the release of free 35S was greater in control than in treated cells, although the 3H patterns of depolymerization with this agent were similar. These data demonstrate that IFNgamma inhibits the incorporation of sulfate from extracellular medium into heparan sulfate.     

Cement versus cementless fixation in total knee arthroplasty

A prospectively studied group of 55 uncemented Press Fit Condylar total knee arthroplasties was compared retrospectively with a matched group of 51 cemented Press Fit Condylar total knee arthroplasties at a mean of 10 years after operation. For the cemented group, the pain and function scores improved from 32 and 45 preoperatively to 95 and 77, respectively. For the uncemented group the scores improved from 33 and 50 preoperatively to 93 and 60, respectively. There were 10 revisions in the uncemented group for femoral or tibial aseptic loosening or osteolysis compared with two revisions in the cemented group. Exclusive of problems related to patellar metal backing, survival to revision for aseptic failure or radiographic loosening was 72% in the uncemented group and 94% in the cemented group at 10 years. A significantly higher revision rate was found in the uncemented compared with cemented total knee arthroplasty of the Press Fit Condylar design.