The core histone N termini function independently of linker histones during chromatin condensation.

The relationships between the core histone N termini and linker histones during chromatin assembly and salt-dependent chromatin condensation were investigated using defined chromatin model systems reconstituted from tandemly repeated 5 S rDNA, histone H5, and either native "intact" core histone octamers or "tailless" histone octamers lacking their N-terminal domains. Nuclease digestion and sedimentation studies indicate that H5 binding and the resulting constraint of entering and exiting nucleosomal DNA occur to the same extent in both tailless and intact chromatin arrays. However, despite possessing a normal chromatosomal structure, tailless chromatin arrays can neither condense into extensively folded structures nor cooperatively oligomerize in MgCl(2). Tailless nucleosomal arrays lacking linker histones also are unable to either fold extensively or oligomerize, demonstrating that the core histone N termini perform the same functions during salt-dependent condensation regardless of whether linker histones are components of the array. Our results further indicate that disruption of core histone N termini function in vitro allows a linker histone-containing chromatin fiber to exist in a decondensed state under conditions that normally would promote extensive fiber condensation. These findings have key implications for both the mechanism of chromatin condensation, and the regulation of genomic function by chromatin.

The use of compensators to optimise the three dimensional dose distribution in radiotherapy of the intact breast.

BACKGROUND AND PURPOSE: Dose heterogeneity in tangential breast irradiation has been shown to be as high as 20% and may lead to problems in local control and cosmesis. In this study, dose heterogeneity in three dimensions (3D) in the breast irradiated with wedged tangential beams is assessed and the improvement which can be made by the use of individualised two dimensional (2D) compensators is established. The compensation required is calculated in two ways: (I) by an iterative technique giving a uniform dose on a plane through the isocentre normal to the central axis of each beam, and (II) by inverse planning using an optimisation technique based on simulated annealing. MATERIALS AND METHODS: A total of 17 patients with histologically proven T0-3, N0, N1, M0 breast cancer undergoing breast irradiation following wide local excision, were CT scanned using contiguous 1 cm slices from approximately 2 cm superior to 2 cm inferior of the irradiated volume. The dose distributions are determined using a 3D algorithm that calculates primary and scatter dose separately using a differential scatter air ratio method and corrects both for the presence of heterogeneities. The iterative technique achieves a dose variation of better than 0.5% on the plane through the isocentre with compensation on both beams. Compensation for the lateral beam only is calculated using the optimisation technique in order to minimise the scatter dose to the contralateral breast. The optimisation algorithm minimises the dose variance over the target and sets upper dose limits for the lung and the remainder of the irradiated volume. RESULTS: For the group of patients the average dose heterogeneity in 3D using wedges is 12% (range 8-17%), which reduces to 8% (5-16%) using compensation on a plane and to 5% (4-7%) using the optimisation technique. CONCLUSIONS: Inverse planning is normally used for complex radiotherapy techniques but when applied to tangential breast irradiation, can reduce the dose heterogeneity through the breast as a whole to as little as 4%, with potential benefits in local control and cosmesis.

Nucleosomes, linker DNA, and linker histone form a unique structural motif that directs the higher-order folding and compaction of chromatin.

The compaction level of arrays of nucleosomes may be understood in terms of the balance between the self-repulsion of DNA (principally linker DNA) and countering factors including the ionic strength and composition of the medium, the highly basic N termini of the core histones, and linker histones. However, the structural principles that come into play during the transition from a loose chain of nucleosomes to a compact 30-nm chromatin fiber have been difficult to establish, and the arrangement of nucleosomes and linker DNA in condensed chromatin fibers has never been fully resolved. Based on images of the solution conformation of native chromatin and fully defined chromatin arrays obtained by electron cryomicroscopy, we report a linker histone-dependent architectural motif beyond the level of the nucleosome core particle that takes the form of a stem-like organization of the entering and exiting linker DNA segments. DNA completes approximately 1.7 turns on the histone octamer in the presence and absence of linker histone. When linker histone is present, the two linker DNA segments become juxtaposed approximately 8 nm from the nucleosome center and remain apposed for 3-5 nm before diverging. We propose that this stem motif directs the arrangement of nucleosomes and linker DNA within the chromatin fiber, establishing a unique three-dimensional zigzag folding pattern that is conserved during compaction. Such an arrangement with peripherally arranged nucleosomes and internal linker DNA segments is fully consistent with observations in intact nuclei and also allows dramatic changes in compaction level to occur without a concomitant change in topology.

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.

Fluoride uptake and release characteristics of glass ionomer cements.

The aims of this study were firstly to investigate the fluoride-releasing characteristics of five commercial glass ionomer materials: Ketac Fil, Chemfil Superior, Fuji II LC, Aquacem and Vitrebond. The second aim was to assess the fluoride uptake and subsequent release from the same range of materials. In both tests, ten discs, 6 mm in diameter with a thickness of 1.5 mm, were made for each material. The initial fluoride release was assessed over a 60-day period for all materials. Each disc was immersed in 2 ml of de-ionised water within a plastic vial. The solutions were changed daily up to day 15, and thereafter every 3 and 4 days until the end of the test. All of the materials released measurable amounts of fluoride throughout the test period, with a considerable range on day 1 (15.3-155.2 ppm F). The concentration of fluoride released on the 2nd day fell sharply for all materials (range 6.3-44.3 ppm F). By day 60 all materials continued to release fluoride, albeit to a lesser extent (range 0.9-3.99 ppm F). With regard to the uptake and release of fluoride, a similar protocol was employed, although all samples were immersed in 1 litre of de-ionised water for 60 days to allow the majority of the fluoride to leach out from the materials. The ten pellets for each material were divided into two groups, five samples as control and five samples as test. Each day over a 20-day period all test samples were exposed to a 1000-ppm F solution for 2 min.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of ondansetron with metoclopramide in the prophylaxis of chemotherapy-induced nausea and vomiting: a randomized, double-blind study. International Emesis Study Group.

The efficacy of ondansetron was compared with metoclopramide in the prophylaxis of nausea and vomiting induced by cyclophosphamide greater than or equal to 500 mg/m2 in combination with doxorubicin greater than or equal to 40 mg/m2 or epirubicin greater than or equal to 40 mg/m2. complete anti-emetic protection in the 24 h following chemotherapy was achieved in 26 of 40 (65%) patients treated with ondansetron compared with 17 of 42 (41%) patients treated with metoclopramide. Severe nausea was present in 3% of patients in the ondansetron group and 31% in the metoclopramide group. A worst day analysis of control of emesis and nausea on days 2 and 3 following chemotherapy also demonstrated ondansetron to be more effective than metoclopramide. Both treatments were well tolerated. Ondansetron is more effective as an anti-emetic than metoclopramide in this type of cytostatic therapy.

A phase I/II study of the 5-HT3 antagonist GR38032F in the anti-emetic prophylaxis of patients receiving high-dose cisplatin chemotherapy.

A total of 24 patients who were receiving combination chemotherapy (POMB) including cisplatin at a dose of 100-120 mg/m2 were treated with the 5HT3 antagonist GR38032F (GR) as an anti-emetic prophylaxis. GR was given as a 15-min loading infusion followed by a 24-h infusion at three escalating dose levels of 1, 2 and 4 mg/h. In the first 24 h after commencing treatment, six patients had complete control of nausea and vomiting (CR), two had 1-2 emetic episodes (MR) and five had 3-5 emetic episodes (mR). The major response rate (CR + MR) was thus 35%. Eight responding patients (CR or MR) went on to receive oral GR at 8 or 12 mg t.i.d. for 5 days. In this group there was one CR, one MR, two mRs and four failures (F). There was no evidence of an improved therapeutic effect with increasing dose in either the infusion or the oral section of the study, although numbers were limited in the latter part of the trial. Toxicity was mild, with low-grade headache affecting 25% of patients being the most frequent side effect. Pharmacokinetic data was obtained in six patients at each dose level. There was a progressive rise in clearance with increasing dose, indicating that the kinetics are non-linear. However, there was no evidence of an association between high plasma levels and therapeutic efficacy. GR38032F is well tolerated and has promising single-agent activity in preventing vomiting induced by high-dose cisplatin.

Biochemical abnormality in brush border membrane protein of a patient with congenital microvillus atrophy.

Brush border membrane proteins from a child with congenital microvillus atrophy were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and compared with brush border membrane proteins from normal and disease controls. A predominant band of MW 200K was present in all preparations with the exception of the membrane preparation from the child with congenital microvillus atrophy. Although the band was present in the latter case, it was grossly diminished. The diminished 200K MW band may result in the abnormality of brush border structure. This could account for the involution of the microvilli, the distinguishing feature of congenital microvillus atrophy, and could be the underlying defect in this disease.

Cytoskeleton changes in fibroblast adhesion and detachment.

The organization of the cytoskeleton in several anchorage-dependent fibroblast types has been compared with the pattern of adhesions to a glass substratum which will support either their growth or just their spreading. Components were stained separately for immunofluorescence microscopy using specific antisera against actin, tubulin, and gizzard 10-nm filament protein, and the adhesions were visualized by interference-reflexion microscopy. Of the cytoskeleton features, only stress fibres could be related to the pattern of focal adhesions; as shown before, each focal adhesion lies directly beneath a stress fibre, often near the terminus. Cells spread on fibronectin-treated glass in serum-free medium to arrest the development of focal adhesions, show correspondingly underdeveloped stress fibres. Actin geodesic domes, microtubules, and 10-nm filaments showed no relations with the adhesion pattern. During cell rounding leading to detachment with either EGTA or trypsin, stress fibres begin to disperse in advance of shape change whereas microtubules and 10-nm filaments seem to alter their distribution as a consequence of shape change. We therefore confirm that stress fibres are the cytoskeleton features most directly related to focal adhesions and are cytoskeleton targets for 2 agents which cause rounding and hence detachment. The sequences of events in dispersal of stress fibres by EGTA and by trypsin showed significant differences in detail. With trypsin, fibres higher in the cell and terminating at the cell edge were more sensitive than most basal fibres and, during disintegration, all types of fibre went through an intermediate 'beaded' structure. With EGTA, all stress fibres seemed to be similarly susceptible and the beaded stage was not seen. The implications of these differences for our understanding of the mechanisms of dispersal of stress fibres are discussed.