Amino acid sequence of the N-terminal domain of calf thymus histone H2A.Z.

The minor histone H2A subtype, H2A.Z, has been purified to homogeneity from calf thymus and subjected to automated Edman degradation. The sequence of the first 30 amino acids possesses only 60% homology with major H2A subtypes of the same tissue. This sequence difference is more extreme than that exhibited between evolutionarily distant major H2A subtypes. However, an analysis of secondary structure reveals that H2A.Z and major H2A subtypes exhibit the same general topographical features within their N-terminal domains.

Pharmaceutical design of a novel colon-targeted delivery system using two-layer-coated tablets of three different pharmaceutical formulations, supported by clinical evidence in humans.

Drug delivery systems to the colon are being actively investigated in order to develop oral preparations of peptides and treat local colonic diseases. However, it is difficult to ensure that an oral preparation disintegrates specifically in the human colon. To make a colonic delivery system practical for medical use, in vitro testing methods need to be established in order to determine the specifications of the preparations. To achieve this objective, three pharmaceutical preparations, designed to have different tablet disintegration times, were used to examine three buffers in seven combinations intended to simulate pH changes in the stomach, small intestine, and colon of humans. To validate the in vitro methodology, furthermore, the fate of all the formulations was examined in the gastrointestinal (GI) tract of healthy volunteers. A three-way crossover trial by scintigraphy revealed that the three formulations--in spite of presenting different in vitro tablet disintegration profiles--have comparable transit profiles and excellent colon-targeting properties in the human gastrointestinal tract regardless of gender and age. These facts strongly suggest that this novel delivery system may be useful for the delivery of drugs to the human colon.

The high photoactivity of m-THPC in photodynamic therapy. Unusually strong retention of m-THPC by RIF-1 cells in culture.

5,10,15,20-Tetra(m-hydroxyphenyl)chlorin (m-THPC, Foscan) is an extremely powerful photosensitizer showing up to 200 times the photodynamic activity of Photofrin in patients, in terms of drug/light dose. The influence of treatment conditions on the photodynamic efficacy of m-THPC has been compared to polyhematoporphyrin (PHP), a Photofrin equivalent, and a cationic pyridinium zinc (II) phthalocyanine (PPC), using the RIF-1 cell line. As predicted, the presence of serum during sensitizer incubation reduced the photodynamic efficacy of all three sensitizers. However, the presence of serum during the illumination period only had an inhibitory effect with PHP and PPC but not m-THPC. Quantification of the intracellular levels of sensitizer revealed that this was due to the efflux of PPC and PHP but not m-THPC into the medium, suggesting that m-THPC is tightly sequestered on entering the cell. This may partially explain the high efficacy of m-THPC in clinical photodynamic therapy and also highlights the importance not only of incubation conditions but also illumination conditions when in vitro comparisons are performed.

Decreased efficiency of trypsinization of cells following photodynamic therapy: evaluation of a role for tissue transglutaminase.

Identifying the cellular responses to photodynamic therapy (PDT) is important if the mechanisms of cellular damage are to be fully understood. The relationship between sensitizer, fluence rate and the removal of cells by trypsinization was studied using the RIF-1 cell line. Following treatment of RIF-1 cells with pyridinium zinc (II) phthalocyanine (PPC), or polyhaematoporphyrin at 10 mW cm-2 (3 J cm-2), there was a significant number of cells that were not removed by trypsin incubation compared to controls. Decreasing the fluence rate from 10 to 2.5 mW cm-2 resulted in a two-fold increase in the number of cells attached to the substratum when PPC used as sensitizer; however, with 5,10,15,20 meso-tetra(hydroxyphenyl) chlorine (m-THPC) there was no resistance to trypsinization following treatment at either fluence rate. The results indicate that resistance of cells to trypsinization following PDT is likely to be both sensitizer and fluence rate dependent. Increased activity of the enzyme tissue-transglutaminase (tTGase) was observed following PPC-PDT, but not following m-THPC-PDT. Similar results were obtained using HT29 human colonic carcinoma and ECV304 human umbilical vein endothelial cell lines. Hamster fibrosarcoma cell (Met B) clones transfected with human tTGase also exhibited resistance to trypsinization following PPC-mediated photosensitization; however, a similar degree of resistance was observed in PDT-treated control Met B cells suggesting that tTGase activity alone was not involved in this process.

A comparative study of the cellular uptake and photodynamic efficacy of three novel zinc phthalocyanines of differing charge.

Three novel substituted zinc (II) phthalocyanines (one anionic, one cationic and one neutral) were compared to two clinically used photosensitizers, 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (m-THPC) and polyhematoporphyrin (PHP), as potential agents for photodynamic therapy (PDT). Using the RIF-1 cell line, photodynamic efficacy was shown to be related to cellular uptake. The cationic phthalocyanine (PPC, pyridinium zinc [II] phthalocyanine) had improved activity over the other two phthalocyanines and slightly improved activity over PHP and m-THPC. The initial subcellular localization of each photosensitizer was dependent upon the hydrophobicity and plasma protein binding. The phthalocyanines had a punctate distribution indicative of lysosomes, whereas m-THPC and PHP had a more diffuse cytoplasmic localization. A relocalization of phthalocyanine fluorescence was observed in some cases following low-level light exposure, and this was charge dependent. The anionic phthalocyanine (TGly, tetraglycine zinc [II] phthalocyanine) relocalized to the nuclear area, the localization of the hydrophobic phthalocyanine (TDOPc, tetradioctylamine zinc [II] phthalocyanine) was unchanged, whereas the distribution of the cationic phthalocyanine (PPC) became more cytoplasmic. This suggests that relocalization following low-level irradiation is a critical factor governing efficacy, and a diffuse cytoplasmic distribution may be a determinant of good photodynamic activity.

The induction of apoptosis by a positively charged methylene blue derivative.

Identifying the cellular responses to photodynamic therapy (PDT) is important if the mechanisms of cell death are to be fully understood. PDT with a methylene blue analog DO15 yielded mitochondrial photodamage whilst membrane and lysosomal integrity were maintained. Apoptosis was detected using the DNA stain HO342, by the appearance of 50 kb fragments and by DNA ladder formation. The release of mono- and oligonucleosomes was further quantified using an ELISA protocol. Large DNA fragments were observed immediately following illumination, and nucleosomes were detected at 1-2 h post-treatment. Increasing the dose 4-fold accelerated the apoptotic response to PDT. This is the first report of a thiazine photosensitiser inducing apoptosis and is consistent with recent proposals suggesting that release of mitochondrial components may play an important role in the mechanism of cell death.

The characterisation of three substituted zinc phthalocyanines of differing charge for use in photodynamic therapy. A comparative study of their aggregation and photosensitising ability in relation to mTHPC and polyhaematoporphyrin.

Three substituted zinc (II) phthalocyanines (one anionic, one cationic and one hydrophobic) have been compared to two clinically used photosensitisers, 5,10,15,20-tetra (m-hydroxyphenyl) chlorin (mTHPC) and polyhaematoporphyrin (PHP), as potential agents for photodynamic therapy (PDT). Oxygen-consumption experiments, performed to follow the photo-oxidation of tryptophan, histidine and bovine serum albumin (BSA), suggest that the anionic phthalocyanine is the most efficient photosensitiser. The efficacy of BSA oxidation is much greater than that of tryptophan or histidine, which is partly due to monomerisation of the sensitisers upon binding to BSA. Spectra recorded in aqueous solution reveal that all five compounds are highly aggregated, but monomerisation is induced upon the addition of BSA or methanol. Using a range of methanol-buffer solutions, the aggregation state has been directly related to the efficacy of tryptophan photo-oxidation with maximal rates of oxidation achieved when the sensitiser is monomeric. Using erythrocytes as a simple membrane model, the efficacy of each sensitiser exhibits a different trend from that predicted by oxygen-consumption experiments. The anionic phthalocyanine is the least effective at photohaemolysis, whereas the cationic and hydrophobic phthalocyanines have improved activity over PHP and mTHPC.

Consumer affairs and the valuation of safety.

An appraisal of past work on the value of a statistical life has been conducted for the purpose of assisting decisions in the consumer safety sector. Although contingent valuation is nowadays often cited as the preferred approach for valuing the dominant, human cost, element of safety, a range of other valuation techniques is recognised and used, both in Britain and elsewhere. It is argued that all valuation techniques entail considerable uncertainty for a variety of reasons, some methodological and some attributable to the fact that the value of safety is an ill-defined and fuzzy concept for the individual. In view of this, plus the need to appeal to a diverse constituency, it is proposed that the value of consumer safety be based upon a broad appreciation of the outcomes from the various techniques which exist, rather than pinning all hopes upon any single methodology. Furthermore, in the interests of sound consumer risk management practice and public credibility, it is recommended that uncertainty be openly acknowledged by expressing the value of safety as a range rather than a point value. The range proposed is from Pound Sterling 1 million to Pound Sterling 10 million, with the expectation that for most consumer applications values used might lie within a narrower bandwidth from about Pound Sterling 2 million to Pound Sterling 4 million. It is suggested that the specification of a range, while not everyone's nor every sector's preferred approach, nonetheless has a number of useful attributes. These include endowing decision makers with a degree of flexibility in arriving at safety decisions, considered to be an important issue within the consumer safety sphere where complex contextual factors frequently arise, and which are arguably best handled by a thoughtful and sensitive approach and negotiation, rather than by the application of any more formulaic recipe.

Long-term observation of the effect of peripheral nerve injury in neonatal and young rats.

The purpose of this study was to observe functional recovery and motoneuron death after nerve transection-and-repair in neonatal versus young animals. One hundred nine Lewis rats underwent posterior tibial nerve transection-and-repair at 6 or 22 days of age. Fifty-two and fifty-seven nerves at the 6- and 22-day times were used for endpoint analysis at 1, 3, 10, and 14 months. These assessments included serial functional walking track analysis, electrophysiologic studies, muscle mass evaluation, motoneuron counts with retrograde horseradish peroxidase tracing, and histologic and morphometric nerve analysis. Walking track analysis and nerve conduction velocity indicated significantly poorer functional regeneration in the 6-day-old group than in the 22-day-old group. Muscle mass in the 6-day-old group did not recover as well as in the 22-day-old group. Motoneuron numbers stained with horseradish peroxidase were less in the 6-day-old group than in the 22-day-old group. In contrast, morphometric analysis did not reach significance. This study suggests that the same nerve injury sustained in a neonatal rat is less likely to demonstrate functional recovery than one sustained in a young rat.

Characterization of a microprocessor-controlled tubular multiple metered dose inhaler aerosol generator for inhalation exposures of pharmaceuticals.

A microprocessor-controlled tubular multiple metered dose inhaler (MDI) aerosol generator was constructed for the delivery of pharmaceutical aerosols to inhalation chambers. The MDIs were mounted in four cassettes containing one to four MDIs on a stepped end plate. The MDIs in each cassette were pneumatically activated at intervals that were controlled by the microprocessor. The cassettes permitted easy replacement of each set of MDIs with a fresh set of MDIs whenever necessary. Aerosol concentration was controlled by varying the number of active MDIs in each cassette and the frequency of activations per minute of each row. Aerosol from the MDIs flowed along the long axis of the tube, which provided a path length sufficient to diminish impaction losses. Using a light-scattering device to monitor the aerosol concentration, the pulsatile output from the MDIs in the cassettes was demonstrated to be adequately damped out provided that the dilution/mixing/aging chamber exceeded 3 ft in length. The tube diameter selected was the minimum compatible with mounting the required number of MDIs so that the linear velocity of the aerosol was adequate to efficiently transport the aerosol out of the dilution chamber. Aerosol concentration and particle size data were recorded for a nose-only rodent exposure chamber. Reproducible aerosol concentrations ranging from 0.03 to 0.6 mg/L were generated. Particle sizes ranged from 2- to 3-microm mass median aerodynamic diameter. Thus, the aerosol generated was within the size range suitable for inhalation exposures.

Playground injuries: a scientific appraisal of popular concerns.

The widespread public concern in Britain over childhood injuries arising from playground accidents is examined using a new national data base on leisure accidents managed by the Department of Trade and Industry. Popular beliefs about the level of risk, the nature and cause of injury, and remedial measures are found to be unsupported by the analysis. Playground risks are comparatively low; accident causes are diverse and most involve long bone injuries and not head injuries as has been widely reported; and the cost of some popular risk reduction measures would seem to be incommensurate with the reasonably-anticipated risk reduction which they might afford. It is suggested that for those who wish to reduce risk further, consideration should be given to increasing public awareness of the many causes of risk, of the opportunities which the individual has to control risk, and of the inevitability of residual risk.

Some observations on waterslide injuries.

STUDY OBJECTIVE: To investigate the causes of a series of interperson collisions on a waterslide resulting in serious injury, and to assess adequacy of control systems and legal liability. METHOD: Site investigation of operating characteristics including a traffic light controlled system, organisational safety culture, and user behaviour. RESULTS: Despite a battery of safety features including closed circuit TV, citizen's band radios, a traffic light controlled system, part time supervision, and warning notices, the system was found to be inherently "unsafe" as operated. It placed a small but significant percentage of users in a hazardous situation whereby consecutive riders could collide with each other while in the flume even when conforming with all instructions. The realised risk might have been even higher were not staff and users adopting their own precautionary measures. CONCLUSIONS: The risk to waterslide users, even the majority observing the safety rules, was questionably high, and facility operators were vulnerable to a court ruling that they had failed in their duty of care. It is suggested that waterslide operators elsewhere, who are wholly or partially reliant on traffic light controlled systems, should carefully scrutinise the safety of their operating systems. Members of the public who use these facilities would, in any event, be well advised to remain vigilant whatever the perceived adequacy of the safety system in operation.

Affinity chromatography and affinity labeling of rat liver succinyl-CoA synthetase.

Succinyl-CoA synthetase has been purified to apparent homogeneity from rat liver. The key step in the purification procedure involved adsorption on a GDP dialdehyde (dial-GDP)-adipic dihydrazide-Sepharose 4B column and elution by GDP-Mg2+. Like the pig heart enzyme (Brownie, E. R., and Bridger, W. A. (1972) Can. J. Biochem. 50, 719--724), the rat liver enzyme was an alpha beta heterodimer and only the alpha subunit was phosphorylated by [gamma-32P]GTP. The A 280(0.1%) of the enzyme was determined to be 0.5. Amino acid analyses revealed significant similarities in 50% of the amino acid residues of rat liver and Escherichia coli succinyl-CoA synthetases. However, immunodiffusion analysis failed to reveal any antigenic identity between the two enzymes. Incubation with the affinity label, dial-GDP, in the presence of Mg2+ resulted in a biphasic inactivation of the enzyme. The extent of the rapid phase of inactivation appeared to be related to the extent of dephosphorylation of the enzyme and was prevented by preincubation of the enzyme with GTP-Mg2+. The presence of GDP-Mg2+ in the incubation medium prevented the slow phase of the inactivation and retarded the rapid phase. Dephosphorylated enzyme was approximately 2 orders of magnitude more susceptible to inactivation by dial-GDP than phosphorylated enzyme. Labeling of succinyl-CoA synthetase with [3H]dial-GDP gave a linear relationship between inactivation and incorporation of radioactivity with an extrapolated value of less than 1.2 mol of analog/mol of enzyme at 100% inactivation. The distribution of the label in enzyme that was inactivated 40% was approximately 60% in the alpha subunit and 40% in the beta subunit. Thus, while phosphorylation of the enzyme occurs exclusively in the alpha subunit, the nucleotide binding site appears to include components from both alpha and beta subunits.

5-methylcytosine is localized in nucleosomes that contain histone H1.

We have developed a procedure for purifying highly specific polyclonal antibodies against 5-methylcytosine. These antibodies were used to probe the distribution of 5-methylcytosine among fractionated nucleosomes of mouse cell chromatin. Our results demonstrate that at least 80% of the 5-methylcytosine is localized in nucleosomes that contain histone H1. Native nucleosomes that lack histone H1 or possess high mobility group proteins package DNA that is 1.6- to 2.3-fold undermethylated. We suggest that the preferential association of methylated sequences with histone H1 has functional significance because DNA methylation has been linked to gene inactivation and histone H1 is known to promote chromatin condensation.

Temperature-related variations in the lipid composition of methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains.

Growth of methicillin-susceptible and -resistant strains of Staphylococcus aureus at 28 and 37 degrees C led to changes in the relative amounts of phospholipids recovered. The major phospholipids present in these strains were phosphatidylglycerol, lysophosphatidylglycerol, and diphosphatidylglycerol. Phosphatidylglycerol tended to occur in lower amounts in the resistant strains, but it was the major phospholipid in both methicillin-susceptible and -resistant S. aureus strains. Growth patterns and total lipid concentrations were similar in all strains.

Transferring DNA from electrophoretically resolved nucleosomes to diazobenzyloxymethyl cellulose: properties of nucleosomes along mouse satellite DNA.

Electrophoresis fractionates nucleosomes which possess different protein compositions. We report here a procedure for transferring the DNA components of electrophoretically resolved nucleosomes to diazobenzyloxymethyl cellulose (DBM-paper). Histones are first removed from nucleosome components by electrophoresis in the presence of cetyltrimethylammonium bromide (CTAB), leaving DNA fragments fixed within the original gel as the CTAB salts. The DNA is then converted to the sodium salt, denatured, and electrophoretically transferred to DBM-paper. The overall pattern of DNA on the resulting blot is visualized either by fluorography or by immunoautoradiography. This DNA pattern is then compared with autoradiograms obtained after hybridizing the same blot with specific 32P-labeled probes. Using mouse satellite DNA as a hybridization probe, we illustrate the above techniques and demonstrate that nucleosomes carrying satellite sequences are compositionally heterogeneous. The procedures described here should also be useful in the analysis of the nucleic acid components associated with other nucleoprotein complexes.

Affinity labeling of succinyl-CoA synthetase from Escherichia coli by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate.

The 2'3'-dialdehyde of adenosine 5'-diphosphate, oADP, exhibited the properties of an affinity label with Escherichia coli succinyl-CoA synthetase. Inactivation of this synthetase by oADP followed pseudo-first-order kinetics and was competitively blocked by ADP. The stoichiometry of labeling of the synthetase was 1 mol/mol alpha beta or, extrapolated, 2 mol/mol inactive alpha 2 beta 2 molecule. oADP also exhibited the properties of a substrate, bringing about rapid dephosphorylation of the enzyme. Further specificity of oADP was demonstrated in partially inactivated succinyl-CoA synthetase by selective inhibition of the succinate in equilibrium succinyl-CoA exchange reaction, in comparison to the CoA in equilibrium succinyl-CoA exchange reaction. Modification of the synthetase by oADP resulted in cross-linking of the enzyme, casting uncertainty over the subunit binding site for ADP. Modification of the synthetase by ADP-2'-semialdehyde occurred at a faster rate than that by oADP but exhibited biphasic inhibitor concentration dependence and did not exhibit saturability.

Cumulative trauma disorders: An overview of the problem.

Cumulative trauma disorders-which result from highly-repetitive job activities, sustained loads, and exposure to vibratory forces-are rapidly spreading through the industrialized world. As their prevalence increases, so do the economic and social consequences. This paper provides an overview of the problem to illustrate the fact that addressing the rising incidence of CTDs requires a multidisciplinary approach involving research and programmatic efforts directed on many fronts, most notably medicine, engineering, management, occupational health, and government. After a review of the diagnosis and treatment of cumulative trauma disorders is presented, the importance of ergonomic analysis for identifying workers at risk is discussed. Some prevention strategies currently being implemented in workplaces around the country are also described. Finally, we raise important industry and public policy issues which need to be confronted if the incidence of CTDs is to be reduced and costs of the workers' compensation system contained.

Nerve crush injuries--a model for axonotmesis.

Nerve crush is a commonly used experimental model in the rat; however, a standard method of inducing this injury has not been defined. This study examined six crush techniques that are frequently used and characterized the subsequent nerve injury. Five types of nerve crush using a No. 5 jeweler's forceps and a sixth using a 30-s single crush with a serrated hemostat were studied in the posterior tibial nerve of the Lewis rat. Regeneration was evaluated using serial walking track assessments at 1, 2, 4, 6, and 8 weeks postoperatively. Nerve conduction studies and histological examination were performed at 2 days, 2 weeks, and 8 weeks. Blood-nerve barrier breakdown was observed at 2 days and recovered by 2 weeks. By 4 weeks normal walking track patterns were obtained in all groups. A pattern of Wallerian degeneration and axonal regeneration was noted at 2 weeks, with histological recovery in all groups by 8 weeks. Nerve crush, induced by any of the six methods tested, was similar and provides a reliable model of axonotmesis.

The effect of age on peripheral motor nerve function after crush injury in the rat.

OBJECTIVE: To determine the ontogeny of functional recovery after peripheral nerve crush injury. DESIGN: Comparative study in rats of varying ages. MATERIAL AND METHODS: Sixty-second crush injury was performed on the left posterior tibial nerve. Control animals underwent either nerve transection or sham procedure. Nerve function was evaluated 2, 4, and 8 weeks following injury by walking track analysis. Print length ratio (PLR), (ratio of normal right-sided print length to experimental left-sided print length), was used to evaluate functional recovery. MEASUREMENTS AND MAIN RESULTS: Two weeks after crush injury, adult rats experienced significantly greater functional impairment than both 4-day-old and 3-week-old animals (p < 0.05). Four weeks after injury, the difference in function between 4-day-old and adult rats and between 3-week-old and adult rats became insignificant. Complete recovery had been achieved by 8 weeks in all groups. CONCLUSIONS: These results demonstrate faster functional recovery after nerve injury in immature rats than in adults.