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).     

Pathway engineering for the production of aromatic compounds in Escherichia coli

The standard enzymatic assay for quantification of D-sorbitol in plasma was adapted to the automatic analyzer Cobas Mira S. In the assay, NAD (reagent) in the presence of sorbitoldehydrogenase (SDH; start reagent) converts D-sorbitol to fructose with formation of NADH, which was detected automatically as the difference between the first and last readings at 340 nm. The sample blank values for each specimen were subtracted to exclude both endogenous D-sorbitol and sugars, which also react as substrates for SDH. The method is simple, rapid (40 samples/h), precise down to endogenous concentrations (coefficient of variation < 5%; limit of determination: 0.38 mg/L) and linear up to 100 mg/L. Samples with higher D-sorbitol concentrations were estimated after dilution. The method was used to measure disposition curves of sorbitol in volunteers after a single intravenous dose of 0.8 g sorbitol.     

Protein kinase A activators inhibit agonist induced prostaglandin production in human amnion

Prostaglandin (PG) production by human amnion has been postulated to have a role in the onset of labor. Previous work by ourselves and others has demonstrated that oxytocin, phorbol esters and epidermal growth factor (EGF) increase PGE2 production in human amnion cells by activation of the Phospholipase C/Protein Kinase C (PKC) cascade system. The present study was undertaken to determine the effect of prior activation of the Adenylate Cyclase cascade system upon subsequent stimulation of PGE2 production by oxytocin, phorbol 12-myristate-13-acetate (PMA) or EGF in amnion cells and membrane discs. Isoproterenol, forskolin and dibutyryl cyclic adenosine monophosphate (dbcAMP) were utilized to activate the Adenylate Cyclase system at the receptor, enzyme and second messenger level. In control amnion cells, oxytocin, PMA and EGF each provoked dose dependent increases in PGE2 production. In cells preincubated with dbcAMP, forskolin or isoproterenol, agonist stimulated PGE2 production was markedly (50-90%) inhibited (p < 0.01). Inhibition was dose dependent upon preincubator concentrations. Maximal inhibition by adenylate cyclase activators occurred with 2-4 h of preincubation. In membrane discs, forskolin preincubation also inhibited oxytocin, PMA and EGF stimulation of PGE2 production. Activation of the Adenylate Cyclase system in human amnion cells or membrane discs inhibits the subsequent action of potent stimulators of PGE2 production in human amnion.     

Laparoscopic surgery of adrenal glands: indications and limits

Over the last four years it has been demonstrated that laparoscopy can be used successfully for adrenalectomy, providing certain advantages over conventional open surgery. The aim of this study was to determine the indications for laparoscopic approach in adrenal surgery. From June 1994 to June 1996 laparoscopic transabdominal flank approaches were proposed in patients with a unilateral 8 cm or less, non-malignant tumors of the adrenal gland. For tumors under 4 cm in diameter only secreting tumors were removed. Among 77 patients requiring ablation of the adrenal gland, 50 (65%) underwent a laparoscopic procedure: 29 Conn adenomas, 10 Cushing adenomas, 6 Pheochromocytomas, 4 incidentalomas. One patient had Cushing's disease and underwent bilateral resection. Mean tumor size was 26 mm (7-75 mm). Malignancy was demonstrated in 2 tumors: one cortisone secreting tumor and one leiomyosarcoma. Conversion was required in 4 cases (8%). Mean operative time for unilateral adrenalectomies was 147 minutes (50-300'). There were no deaths. Morbidity included: one hemorrhage via the trocar orifice requiring reoperation, one infarction of the spleen which regressed spontaneously, one parietal hematoma, and one case of phebitis of the lower limb. The endocrinopathy was successfully cured in all patients with secreting tumors. The 27 other patients underwent open adrenalectomy. Laparoscopic approach was not proposed due to suspected malignancy in 13 cases, previous surgery in 8 cases and multiple, bilateral and/or extra adrenal tumors in 6 cases. Laparoscopic approach to the adrenal gland is the procedure of choice in patients with Conn adenomas, Cushing adenomas and in most cases of pheochromocytomas. It is not indicated for malignant and large tumor (> 8 cm). Currently two-thirds of our patients requiring and adrenalectomy are operated laparoscopically.     

Laparoscopically treated extrauterine pregnancy

During a period of 12 months 55 women were treated for 56 ectopic pregnancies. Forty of the 56 (71%) procedures were performed laparoscopically. The duration of operation was 74 minutes when a conservative procedure was used, and 83 minutes where a salpingectomy was performed. The median duration of a diagnostic laparoscopy followed by laparotomy in 12 women was 83 minutes. Four women (13%) had persistent trophoblast, which necessitated a second operation. Two patients had a second laparoscopy because of lower abdominal pain, but did not need further treatment. Median hospitalization time (including diagnosis and second procedures) for the laparoscopically treated women was three days (range one to 16 days).     

The role in cell binding of a beta-bend within the triple helical region in collagen alpha 1 (I) chain: structural and biological evidence for conformational tautomerism on fiber surface

Dystrophin is a plasma membrane-associated cytoskeletal protein of the spectrin superfamily. The dystrophin cytoskeleton has been first characterized in muscle. Muscular 427 kDa dystrophin binds to subplasmalemmal actin filaments via its amino-terminal domain. The carboxy-terminus of dystrophin binds to a plasma membrane anchor, beta-dystroglycan, which is associated on the external side with the extracellular matrix receptor, alpha-dystroglycan, that binds to the basal lamina proteins laminin-1, laminin-2, and agrin. In the muscle, the dystroglycan complex is associated with the sarcoglycan complex that consists of several glycosylated, integral membrane proteins. The absence or functional deficiency of the dystrophin cytoskeleton is the cause of several types of muscular dystrophies including the lethal Duchenne muscular dystrophy (DMD), one of the most severe and most common genetic disorders of man. The dystrophin complex is believed to stabilize the plasma membrane during cycles of contraction and relaxation. Muscular dystrophin and several types of dystrophin variants are also present in extramuscular tissues, e.g. in distinct regions of the central nervous systems including the retina. Absence of dystrophin from these sites is believed to be responsible for some extramuscular symptoms of DMD, e.g. mental retardation and disturbances in retinal electrophysiology (reduced b-wave in electroretinograms). The reduced b-wave in electroretinograms indicated a disturbance of neurotransmission between photoreceptors and ON-bipolar cells. At least two different dystrophin variants are present in photoreceptor synaptic complexes. One of these dystrophins (Dp260) is virtually exclusively expressed in the retina. In the neuroretina, dystrophin is found in significant amounts in the invaginated photoreceptor synaptic complexes. At this location dystrophin colocalizes with dystroglycan. Agrin, an extracellular ligand of alpha-dystroglycan, is also present at this location whereas the proteins of the sarcoglycan complex appear to be absent in photoreceptor synaptic complexes. Dystrophin and dystroglycan are located distal from the ribbon-containing active synaptic zones where both proteins are restricted to the photoreceptor plasma membrane bordering on the lateral sides of the synaptic invagination. In addition, some neuronal profiles of the postsynaptic complex also contain dystrophin and beta-dystroglycan. These profiles appear to belong at least in part to projections of the photoreceptor terminals into the postsynaptic dendritic complex. In view of the abnormal neurotransmission between photoreceptors and ON-bipolar cells in DMD patients the dystrophin/beta-dystroglycan-containing projections of photoreceptor presynaptic terminals into the postsynaptic dendritic plexus might somehow modify the ON-bipolar pathway. Another retinal site associated with dystrophin/beta-dystropglycan is the plasma membrane of Müller cells where dystrophin/beta-dystroglycan appear to be present at particular high concentrations. At this location the dystrophin/dystroglycan complex may play a role in the attachment of the retina to the vitreous, and, under pathological conditions, in traction-induced retinal detachment.     

Spontaneous erythroid colony formation as the clue to an underlying myeloproliferative disorder in patients with Budd-Chiari syndrome or portal vein thrombosis

Results from this laboratory have shown that bone metabolism is directly related to extracellular pH and that high concentrations of tobramycin released from impregnated polymethylmethacryrate (PMMA) beads has pH-dependent toxic effects on bone. In the present study, beneficial effects of calcium hydroxide-impregnated PMMA were investigated regarding tobramycin toxicity and bone metabolism in chick embryo tibiae in vitro. Also using Ca(OH)2 as a pH regulator, the antibiotic efficacy of tobramycin-impregnated PMMA was evaluated with respect to inhibition of Staphylococcus aureus growth. When Ca(OH)2 was added to PMMA beads containing tobramycin, the beads released hydroxyl and calcium ions into the culture medium and released more antibiotic than beads containing only tobramycin. Bone metabolism (glycolysis, total protein synthesis, and collagen synthesis) was enhanced by Ca(OH)2-impregnated beads with or without tobramycin. Additionally, bacterial growth was inhibited more strongly when S. aureus was incubated with tobramycin- and Ca(OH)2-impregnated PMMA disks than with disks containing only tobramycin. This study demonstrates the feasibility of adding Ca(OH)2 to tobramycin-impregnated PMMA beads as a regulator of local pH and a promoter of bone metabolism for protection of bone when high concentrations of tobramycin are used to treat osteomyelitis. It also suggests that lower concentrations of antibiotic may be effective if Ca(OH)2 and tobramycin are administered simultaneously.     

Synthesis and release of ATP by soluble mitochondrial F1 in complex with its inhibitor protein during dimethylsulfoxide-water transitions

Soluble mitochondrial F1 and F1 in complex with the natural ATPase inhibitor protein (F1-IP) catalyze the spontaneous synthesis of [gamma-32P]ATP from medium [32P]phosphate and enzyme-bound ADP when incubated in media with dimethylsulfoxide (Me2SO); under these conditions, the synthesized [gamma-32P]ATP is not released into the media, it remains tightly bound to the enzymes [Gómez-Puyou, A., Tuena de Gómez-Puyou, M. & de Meis, L. (1986) Eur. J. Biochem. 159, 133-140]. Some of the characteristics of the synthesized [gamma-32P]ATP were studied in F1 and F1-IP (ATPase activities of 70 and 1-3 micromol x min(-1) x mg(-1), respectively). In Me2SO media, gamma-phosphate of synthesized ATP in F1 or F1-IP exchanges with medium phosphate. From the rates of the exchange reaction, the half-times for hydrolysis of the synthesized ATP in F1 and F1-IP were calculated: 45 min and 58 min for F1 and F1-IP, respectively. The course that synthesized [gamma-32P]ATP follows after dilution of the Me2SO synthetic mixture with aqueous buffer was determined. After dilution, the half-life of synthesized ATP in F1 was less than 1 min. In F1-IP, ATP was also hydrolyzed, but at significantly lower rates. In F1-IP, dilution also produced release of the synthesized [gamma-32P]ATP. This was assayed by the accessibility of [gamma-32P]ATP to hexokinase. About 25% of [gamma-32P]ATP synthesized in F1-IP, but not in F1, was released into the media after dilution with aqueous buffer that contained 20 mM phosphate. Release of tightly bound ATP required the binding energy of phosphate and solvation of F1-IP, however, the particular kinetics of F1-IP were also central for medium ATP synthesis in the absence of electrochemical H+ gradients.