Headteachers' and chairs of governors' perspectives on adolescent obesity and its prevention in English secondary school settings.

BACKGROUND: Secondary schools are an important setting for preventing obesity in adolescence. Headteachers and chairs of governors are identified in national guidance as crucial stakeholders for school-based preventative action. Despite this, their views remain unexplored and unrepresented. METHODS: A sequential mixed method study was conducted. Semi-structured interviews were undertaken with a purposive sample of 22 secondary school headteachers and chairs of governors in England. Data were thematically analysed and informed the development of a descriptive cross-sectional survey, completed by 127 participants from the same population. RESULTS: Unhealthy dietary and sedentary behaviours were viewed as a more significant problem than adolescent obesity. Obesity was perceived as complex and multi-causal, and a range of stakeholders were deemed to have responsibility for its prevention, most notably parents. Support was identified for the role of secondary schools, although this was not an explicit priority and extensive internal and external barriers exist, which hinder preventative action. CONCLUSIONS: Whilst secondary school settings in England remain an important setting for the prevention of adolescent obesity, it is crucial for policy makers and public health professionals to recognize the factors affecting school leaders' ability and willingness to contribute to this agenda.

Measurement of the Vector and Tensor Asymmetries at Large Missing Momentum in Quasielastic (e[over →],e

We report the measurement of the beam-vector and tensor asymmetries A_{ed}^{V} and A_{d}^{T} in quasielastic (e[over →],e^{'}p) electrodisintegration of the deuteron at the MIT-Bates Linear Accelerator Center up to missing momentum of 500 MeV/c. Data were collected simultaneously over a momentum transfer range 0.1

A FRET-based method for monitoring septin polymerization and binding of septin-associated proteins.

Much about septin function has been inferred from in vivo studies using mainly genetic methods, and much of what we know about septin organization has been obtained through examination of static structures in vitro primarily by electron microscopy. Deeper mechanistic insight requires real-time analysis of the dynamics of the assembly of septin-based structures and how other proteins associate with them. We describe here a Förster resonance energy transfer (FRET)-based approach for measuring in vitro the rate and extent of filament formation from septin complexes, binding of other proteins to septin structures, and the apparent affinities of these interactions. FRET is particularly well suited for interrogating protein-protein interactions, especially on a rapid timescale; the spectral change provides an unambiguous indication of whether two elements within the system under study are associating and serves as a molecular-level "ruler" because it is very sensitive to the separation between the donor and acceptor fluorophores over biologically relevant distances (≤10nm). The necessary procedures involve generation of appropriate cysteine-less and single cysteine-containing septin variants, expression and purification of the heterooctameric complexes containing them, efficient labeling of the purified complexes with desired fluorophores, fluorimetric measurement of FRET, and appropriate safeguards and controls in data acquisition and analysis. Our methods can be used to interrogate the effects of buffer conditions, small molecules, and septin-binding proteins on septin filament assembly or stability; determine the effect of alternative septin subunits, mutational alterations, or posttranslational modifications on assembly; and, delineate the location of septin-binding proteins.

The risk of foot ulceration in people with diabetes screened in community settings: findings from a cohort study.

BACKGROUND: Annual foot checks are recommended in patients with diabetes mellitus (DM) to identify those at risk of foot ulceration. Systematic reviews have found few studies evaluating the predictive value of tests in community-based diabetic populations. AIM: To quantify the predictive value of clinical risk factors in relation to foot ulceration in a community population. METHODS: A cohort of 1192 people with diabetes receiving care in community settings was recruited and a screening procedure, covering symptoms, signs and diagnostic tests was conducted at baseline. At an average 1-year follow-up patients who developed a foot ulcer were identified by an independent blind assessor. Multivariable analysis was performed to identify clinical predictors of foot ulceration. FINDINGS: The incidence of foot ulceration was 1.93% [95% confidence interval (CI) 1.27-2.89). Three time-independent clinical predictors with five factors were selected: previous amputation [odds ratio (OR) 14.7, 95% CI 3.1-69.5), use of insulin before 3 months with inability to distinguish between cool and cold temperatures (OR 2.97, 95% CI 1.9-4.5) and failure to obtain at least one blood pressure reading for the calculation of ankle-brachial index with the failure to feel touch with a 10-g monofilament (OR 1.7, 95% CI 1.3-2.2). INTERPRETATION: Recommendations for annual diabetic foot check in low-risk, community-based patients should be reviewed as absolute events of ulceration are low. The accuracy of foot risk assessment tools to predict ulceration requires evaluation in randomized controlled trials with concurrent economic evaluations.

Precise measurement of deuteron tensor analyzing powers with BLAST.

We report a precision measurement of the deuteron tensor analyzing powers T(20) and T(21) at the MIT-Bates Linear Accelerator Center. Data were collected simultaneously over a momentum transfer range Q=2.15-4.50 fm(-1) with the Bates Large Acceptance Spectrometer Toroid using a highly polarized deuterium internal gas target. The data are in excellent agreement with calculations in a framework of effective field theory. The deuteron charge monopole and quadrupole form factors G(C) and G(Q) were separated with improved precision, and the location of the first node of G(C) was confirmed at Q=4.19±0.05 fm(-1). The new data provide a strong constraint on theoretical models in a momentum transfer range covering the minimum of T(20) and the first node of G(C).

Charge form factor of the neutron at low momentum transfer from the 2H-->(e-->,e'n)1H reaction.

We report new measurements of the neutron charge form factor at low momentum transfer using quasielastic electrodisintegration of the deuteron. Longitudinally polarized electrons at an energy of 850 MeV were scattered from an isotopically pure, highly polarized deuterium gas target. The scattered electrons and coincident neutrons were measured by the Bates Large Acceptance Spectrometer Toroid (BLAST) detector. The neutron form factor ratio GEn/GMn was extracted from the beam-target vector asymmetry AedV at four-momentum transfers Q2=0.14, 0.20, 0.29, and 0.42 (GeV/c)2.

-Measurement of the proton's electric to magnetic form factor ratio from 1H(over -->)(e(over -->),e'p).

We report the first precision measurement of the proton electric to magnetic form factor ratio from spin-dependent elastic scattering of longitudinally polarized electrons from a polarized hydrogen internal gas target. The measurement was performed at the MIT-Bates South Hall Ring over a range of four-momentum transfer squared Q2 from 0.15 to 0.65 (GeV/c)(2). Significantly improved results on the proton electric and magnetic form factors are obtained in combination with existing cross-section data on elastic electron-proton scattering in the same Q2 region.

Comparison of the metabolism of ethylene glycol and glycolic acid in vitro by precision-cut tissue slices from female rat, rabbit and human liver.

1. The metabolism of [1,2-(14)C]-ethylene glycol and [1,2-(14)C]-glycolic acid was studied in vitro using precision-cut tissue slices prepared from the livers of female Sprague-Dawley rats, New Zealand white rabbits and humans. The time-course for production of metabolites formed from ethylene glycol at concentrations from 3 to 40 mM was determined to compare quantitatively the differences between species in the rates and amounts of formation of glycolic acid, the presumed developmental toxicant of ethylene glycol. The rates of metabolism of glycolic acid to glyoxylic acid at concentrations from 0.05 to 16 mM by liver tissue from the different species were also determined. The apparent V(max)/K(m) for the metabolic conversions of ethylene glycol to glycolic acid and for glycolic acid to glyoxylic acid in liver tissue from the different species were obtained. 2. There were qualitative differences in the metabolic profiles and quantitative differences in the formation of glycolic acid between the mammalian liver systems. There was an average of 10-fold less glycolic acid produced by liver slices from rabbits compared with rats. With the human liver, the formation of glycolic acid was not detectable using tissue from three of four human donors. A low level of glycolic acid was detected in one liver slice incubation from one of the four subjects, but only at one extended time point; glyoxylate was detected with liver slices from all four humans. 3. Liver slices prepared from female Sprague-Dawley rats, female New Zealand White rabbits and three female human subjects all metabolized glycolic acid to glyoxylic acid. Human liver tissue was the most effective at further metabolizing glycolic acid to glyoxylic acid. The ratios of V(max)/K(m), representing the relative clearance of glycolic acid from liver tissue, were approximately 14:9:1 for human, rat and rabbit liver, respectively. 4. Precision-cut liver slices maintained in dynamic organ culture are good predictors of metabolism by liver tissue in vivo. The results of the present study therefore indicate that levels of glycolic acid, if formed in vivo, following exposures to similar concentrations of ethylene glycol, would be lower in humans than in rabbits and rats.

Effects of solvent on DNA adduct formation in skin and lung of CD1 mice exposed cutaneously to benzo(a)pyrene.

The effect of solvent polarity and lipophilicity on DNA adduct formation by polycyclic aromatic hydrocarbons in skin and lung has been studied in CD1 mice exposed cutaneously in vivo to benzo(a)pyrene ( approximately 0.01-7.0 microg/animal) in either tetrahydrofuran or n-dodecane. The nature and amounts of DNA adducts, measured as 7R,8S, 9R-trihydroxy-10S-(N(2)-deoxyguanosyl-3'-phosphate)-7,8,9, 10-tetrahydrobenzo(a)pyrene, in relation to exposure dose and treatment regime was determined by (32)P-postlabelling. In skin DNA there was a linear relationship between exposure dose and adduct formation with both solvents, though the amount of adduct formed was significantly lower from treatment with benzo(a)pyrene in n-dodecane than in tetrahydrofuran. The amounts of adducts measured in skin DNA ranged from 67 amol adducts/microg DNA at the lowest exposure dose of benzo(a)pyrene in n-dodecane to 3.5 fmol adducts/microg DNA (1 adduct in 5 x 10(7) nucleotides to 1 adduct in 9 x 10(5) nucleotides) at the highest dose. In tetrahydrofuran the corresponding levels were 89 amol adducts/microg DNA (1 adduct in 3 x 10(7) nucleotides) to 16.9 fmol adducts/microg DNA (1 adduct in 2 x 10(5) nucleotides). DNA adducts could not be detected in lung tissue following cutaneous treatment of animals with benzo(a)pyrene in n-dodecane. Cutaneous treatment of animals with benzo(a)pyrene in tetrahydrofuran, however, resulted in adducts in lung DNA at a level of 88 amol/microg DNA from exposures only at the highest dose (6.72 microg/animal). The difference in octanol-water partition coefficient, log P(ow) between n-dodecane compared to tetrahydrofuran is considered to be the most likely reason for the reduction in the bioavailability of benzo(a)pyrene and/or its metabolites and hence the degree of genotoxicity in tissues. The results suggest that other paraffinic hydrocarbon solvents may moderate the genotoxicity of polycyclic aromatic hydrocarbons in vivo. The assessment of the genotoxicity in vivo of mixtures of compounds should be carried out on complete mixtures of substances of interest in order to take account of these possible antagonistic or synergistic effects.

Quantitative and qualitative differences in the metabolism of 14C-1,3-butadiene in rats and mice: relevance to cancer susceptibility.

1,3-Butadiene (butadiene) is a potent carcinogen in mice, but not in rats. Metabolic studies may provide an explanation of these species differences and their relevance to humans. Male Sprague-Dawley rats and B6C3F1 mice were exposed for 6 h to 200 ppm [2,3-14C]-butadiene (specific radioactivity [sa] 20 mCi/mmol) in a Cannon nose-only system. Radioactivity in urine, feces, exhaled volatiles and 14C-CO2 were measured during and up to 42 h after exposure. The total uptake of butadiene by rats and mice under these experimental conditions was 0.19 and 0.38 mmol (equivalent to 3.8 and 7.5 mCi) per kg body weight, respectively. In the rat, 40% of the recovered radioactivity was exhaled as 14C-CO2, 70% of which was trapped during the 6-h exposure period. In contrast, only 6% was exhaled as 14C-CO2 by mice, 3% during the 6-h exposure and 97% in the 42 h following cessation of exposure. The formation of 14C-CO2 from [2,3-14C]-labeled butadiene indicated a ready biodegradability of butadiene. Radioactivity excreted in urine accounted for 42% of the recovered radioactivity from rats and 71% from mice. Small amounts of radioactivity were recovered in feces, exhaled volatiles and carcasses. Although there was a large measure of commonality, the exposure to butadiene also led to the formation of different metabolites in rats and mice. These metabolites were not found after administration of [4-14C]-1,2-epoxy-3-butene to animals by i.p. injection. The results show that the species differences in the metabolism of butadiene are not simply confined to the quantitative formation of epoxides, but also reflect a species-dependent selection of metabolic pathways. No metabolites other than those formed via an epoxide intermediate were identified in the urine of rats or mice after exposure to 14C-butadiene. These findings may have relevance for the prediction of butadiene toxicity and provide a basis for a revision of the existing physiologically based pharmacokinetic models.

Reduction of myocardial infarct size after ischemia and reperfusion by the glycosaminoglycan pentosan polysulfate.

Activation of the complement system contributes to the tissue destruction associated with myocardial ischemia/reperfusion. Pentosan polysulfate (PPS), a negatively charged sulfated glycosaminoglycan (GAG) and an effective inhibitor of complement activation, was studied for its potential to decrease infarct size in an experimental model of myocardial ischemia/reperfusion injury. Open-chest rabbits were subjected to 30-min occlusion of the left coronary artery followed by 5 h of reperfusion. Vehicle (saline) or PPS (30 mg/kg/h) was administered intravenously immediately before the onset of reperfusion and every hour during the reperfusion period. Treatment with PPS significantly (p < 0.05) reduced infarct size as compared with vehicle-treated animals (27.5+/-2.9% vs. 13.34+/-2.6%). Analysis of tissue demonstrated decreased deposition of membrane-attack complex and neutrophil accumulation in the area at risk. The results indicate that, like heparin and related GAGs, PPS possesses the ability to decrease infarct size after an acute period of myocardial ischemia and reperfusion. The observations are consistent with the suggestion that PPS may mediate its cytoprotective effect through modulation of the complement cascade.

Studies on the dermal and systemic bioavailability of polycyclic aromatic compounds in high viscosity oil products.

The assessment of skin penetration by viscous oil products is an important element in the risk assessment of these materials where skin contact is likely. Systemic bioavailability (body uptake) is viewed as a good indicator of skin penetration following cutaneous exposures. The results of this study provide quantitative information on the influence of viscosity on the bioavailability of a specific polycyclic aromatic compound (benzo(a)pyrene) in base oils, residual aromatic extracts and bitumens following skin exposures to mice. The materials studied were a base mineral oil (viscosity 32 cSt at 35 degrees C), a 1:1 blend of the mineral base oil and a residual aromatic extract (198 cSt), several residual aromatic extracts (ca. 5000 cSt, 35 degrees C) and a range of bitumens (0.65-69 x 10(6) cSt, 35 degrees C). These were each spiked with 0.1% radiolabelled benzo(a)pyrene, as a representative carcinogenic polycyclic aromatic compound, then used for cutaneous exposures to mice. The results indicate that as viscosity increased in the range ca. 30 to 5000 cSt (base oil to residual aromatic extract) the uptake of the radiolabelled benzo(a)pyrene into blood was reduced by ca. fivefold. Further increases in viscosity from ca. 5000 to 69 x 10(6) cSt (i.e. residual aromatic extract to bitumen) resulted in a further but smaller (ca. twofold) reduction in uptake. The relationship between the amounts of free benzo(a)pyrene measured in blood and viscosity showed the same trend. This trend was also mirrored by the degree of binding of benzo(a)pyrene metabolites to DNA in skin. The findings in mouse skin in vivo indicate that viscosity can significantly affect skin penetration and systemic bioavailability of polycyclic aromatic compound components of oil products. Results obtained with viable human skin in vitro also showed that the bioavailability of benzo(a)pyrene was reduced by the viscosity of the oil product matrix. It is thus necessary to take account of physical properties such as viscosity in the overall risk assessment of viscous oil products, particularly in the case of very viscous materials such as bitumens. The significantly reduced bioavailability of hazardous compounds from undiluted materials is thus an important factor to consider when assessing the risks from dermal exposures.

Development of a carcinogenic potency index for dermal exposure to viscous oil products.

Polycyclic aromatic compounds (PACs) present in oil streams and formulated products are important determinants of possible carcinogenicity. Following dermal exposures the transport of the PACs from oil (the carrier) into the skin is a factor that may affect macromolecular (DNA) adduct formation and thus determine carcinogenicity. We have developed a mathematical model, which describes the flux into the skin for a representative carcinogenic PAC, benzo(a)pyrene. The model is based on measurements of the amount of benzo(a)pyrene bound to skin DNA or blood observed in mouse skin painting studies. The degree of adduct formation from a particular oil product, which we term the Bioavailability Index (BI), was shown to be a function of both the viscosity of the oil product, which affected the transport of the PAC through the carrier, and the aromaticity, which affected the partition of PAC between the carrier and the skin. Literature data were analysed from mouse skin painting studies with mineral oils of known carcinogenicity. A linear relationship was shown between the amount of DNA adduct formation, expressed as alkylation frequency, and the arithmetic product of the total (3-6) ring PAC content and the BI, which we have termed the Carcinogenic Potency Index (CPI). Comparison of literature data on DNA alkylation frequencies for oil products and their carcinogenicity indicated that oils giving rise to an alkylation frequency below a certain threshold (ca. 1 adduct in 10(8) nucleotides) are non-carcinogenic to mouse skin. This threshold level can be translated into a value for the CPI, below which the genotoxic carcinogenic risk arising from skin contact with the oil product is considered to be negligible. The CPI for bitumens is well below this value, being both due to the low BI from bitumen, but more so, due to their low PAC content. For some bitumens diluted with solvents, i.e. cutback-bitumens, the CPI may exceed this value, indicating a possible carcinogenic risk for some of the cutback-bitumens. The main determining factor is the PAC content which is principally determined by the nature of the diluent used.

MRI, quantitative MRI, SPECT, and neuropsychological findings following carbon monoxide poisoning.

Carbon monoxide (CO) poisoning has been shown to result in neuropathologic changes and cognitive impairments due to anoxia and other related biochemical mechanisms. The present study investigated brain-behaviour relationships between neuropsychological outcome and SPECT, MRI, and Quantitative magnetic resonance imaging (QMRI) in 21 patients with CO poisoning. Ninety-three per cent of the patients exhibited a variety of cognitive impairments, including impaired attention, memory, executive function, and mental processing speed. Ninety-five per cent of the patients experienced affective changes including depression and anxiety. The results from the imaging studies revealed that 38% of the patients had abnormal clinical MRI scans, 67% had abnormal SPECT scans, and 67% had QMRI findings including hippocampal atrophy and/or diffuse cortical atrophy evidenced by an enlarged ventricle-to-brain ratio (VBR). Hippocampal atrophy was also found on QMRI. SPECT and QMRI appear to be sensitive tools which can be used to identify the neuropathological changes and cerebral perfusion defects which occur following CO poisoning. Cerebral perfusion defects include frontal and temporal lobe hypoperfusion. Significant relationships existed between the various imaging techniques and neuropsychological impairments. The data from this study indicate that a multi-faceted approach to clinical evaluation of the neuropathological and neurobehavioural changes following CO poisoning may provide comprehensive information regarding the neuroanatomical and neurobehavioural effects of CO poisoning.

N-Acetylheparin pretreatment reduces infarct size in the rabbit.

The ability of the heparin derivative, N-acetylheparin (NHEP) to protect the heart from regional ischemia/reperfusion injury was examined in vivo. NHEP (2 mg/kg i.v.) or vehicle was administered 2 h before occlusion of the left circumflex coronary (LCX) artery. Open-chest, anesthetized rabbits were subjected to 30 min of regional myocardial ischemia followed by 5 h of reperfusion. Myocardial myeloperoxidase activity, membrane attack complex (MAC) deposition and IL-8 generation were assessed in supernatant samples from the area at risk. Infarct size in rabbits pretreated with NHEP (32.5 +/- 3.8%, n = 10) decreased by 41% compared to infarct size in rabbits that received vehicle (55.3 +/- 4.9%, n = 10; p = 0.002). Accumulation of neutrophils within the ischemic region, as assessed by myeloperoxidase activity, declined by 45% (p < 0.05) in AAR from NHEP-treated animals compared to AAR from vehicle-treated animals. Levels of MAC and IL-8 obtained from AAR were less in NHEP-pretreated animals compared to controls. These results suggest that NHEP may protect the myocardium by inhibiting complement activation and subsequent neutrophil infiltration.

Correlation of 32P-postlabelling-detection of DNA adducts in mouse skin in vivo with the polycyclic aromatic compound content and mutagenicity in Salmonella typhimurium of a range of oil products.

The in vivo genotoxic activities in mouse skin of the dimethyl sulphoxide (DMSO) extracts of a range of oil products [residual aromatic extract; untreated heavy paraffinic distillate aromatic extract; mildly refined light naphthenic base oil; bitumen (vacuum residue); high viscosity index base oil obtained by catalytic hydrogenation] were evaluated by 32P-postlabelling DNA analysis. The results of quantitative 32P-postlabelling analyses of epidermal DNA from mice treated with the DMSO extracts showed linear relationships with the total polycyclic aromatic compound (PAC) contents, determined by the Institute of Petroleum method IP 346 and also the 3-6 ring PAC contents, measured by on-line liquid-liquid extraction using flow injection analysis. The 32P-postlabelling data also showed a linear relationship with the mutagenicity indices of these oil products determined in S. typhimurium TA98 using the modified Ames Salmonella microsome test. The in vivo genotoxicity of the DMSO extracts from the oil products was low, judged by 32P-postlabelling analysis of DNA adducts measured in epidermal DNA of treated mouse skin, and ranging from 2 to 723 attomole/microg DNA per mg oil product. The in vivo 32P-postlabelling data from this study are consistent with these materials expressing low genotoxicity in mouse skin in vivo. The DMSO extraction procedure coupled with 32P-postlabelling DNA analysis is useful for ranking the relative genotoxic potency in vivo of a wide range of oil products. In general the trend observed is similar to rankings based on physicochemical measurements of total PAC contents or 3 6 ring PAC contents of the oil products.

The semisynthetic polysaccharide pentosan polysulfate prevents complement-mediated myocardial injury in the rabbit perfused heart.

Pentosan polysulfate (PPS) is a highly sulfated semisynthetic polysaccharide possessing a higher negative charge density and degree of sulfation than heparin. Like other glycosaminoglycans, the structural and chemical properties of PPS promote binding of the drug to the endothelium. Glycosaminoglycans, including heparin, inhibit complement activation independent of an action on the coagulation system. This ability provides a compelling argument for the implementation of this class of compounds in experimental models of cellular injury mediated by complement. The objective of this study was to examine whether PPS could reduce myocardial injury resulting from activation of the complement system. We used the rabbit isolated heart perfused with 4% normal human plasma as a source of complement. Hemodynamic variables were obtained before addition of PPS (0.03 01 mg/ml) and every 10 min after the addition of human plasma. Compared with vehicle-treated hearts, left ventricular end-diastolic pressure was improved at the conclusion of the 60-min protocol in hearts treated with PPS (58.9 +/- 13.6 vs. 15. 2 +/- 4.8 mm Hg). Further evidence as to the protective effects of PPS was demonstrated by decreased creatine kinase release compared with vehicle (86.5 +/- 28.5 U/l vs. 631.0 +/- 124.8 U/l). An enzyme-linked immunosorbent assay for the presence of the membrane attack complex in lymph and tissue samples demonstrated decreased membrane attack complex formation in PPS-treated hearts, which suggests inhibition of complement activation. This conclusion was supported further by the ability of PPS to inhibit complement-mediated red blood cell lysis in vitro. The results of this study indicate that PPS can reduce tissue injury and preserve organ function that otherwise would be compromised during activation of the human complement cascade.

Comparison of immunoaffinity chromatography enrichment and nuclease P1 procedures for 32P-postlabelling analysis of PAH-DNA adducts.

32P-postlabelling analysis for detecting DNA adducts formed by polycyclic aromatic compounds is one of the most widely used techniques for assessing genotoxicity associated with these compounds. In cases where the formation of adducts is extremely low, a crucial step in the analysis is an enrichment procedure for adducts prior to the radiolabelling step. The nuclease P1 enhancement procedure is the most established and frequently used of these methods. An immunoaffinity procedure developed for class specific recognition for polycyclic aromatic hydrocarbon (PAH)-DNA adducts has therefore been compared with the nuclease P1 method for a range of DNA adducts formed by PAHs. The evaluation was carried out with skin DNA from mice treated topically with benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 5-methylchrysene or chrysene. The immobilised antibody had the highest affinity for adducts structurally similar to the BPDE-I-deoxyguanosine adduct ([+/-]-N2-(7r,8t,9r-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-1 0t-yl)-2'-deoxyguanosine) against which the antibody had been raised. Of the PAH-modified DNAs evaluated, the maximum adduct recovery was obtained for DNA containing the BPDE I-deoxyguanosine adduct. With DMBA-modified DNA, the profiles of adducts recovered from the column were similar when the column material was treated either with a digest of DMBA-modified DNA or with 32P-labelled DMBA adducts. I-compounds (endogenous adducts in tissue DNA of unexposed animals), which had similar chromatographic properties to PAH-DNA adducts, were not enriched by the immunoaffinity procedure. Compared to the simple nuclease P1 enhancement procedure, the immunoaffinity methods were lengthier and more labour intensive. Advantages of the immunoaffinity procedure include: specificity, allowing the selective detection of a certain class of adducts: efficient adduct enrichment, providing a viable alternative to other enrichment procedures; adequate sensitivity for model studies and the potential to purify adducts for further characterisation. However, as a general screen for detecting the formation of DNA adducts, the nuclease P1 procedure was viewed as the initial method of choice since it was capable of detecting a wider range of PAH-DNA adducts.

Attenuation of interleukin-8 expression in C6-deficient rabbits after myocardial ischemia/reperfusion.

Neutrophil accumulation and activation of the complement system with subsequent deposition of the cytolytic membrane attack complex (MAC) have been implicated in the pathogenesis of myocardial ischemia/reperfusion injury. The MAC, when present in high concentrations, promotes target cell lysis. However, relatively little is known about the potential modulatory role of sublytic concentrations of the MAC on nucleated cell function in vivo. In vitro studies demonstrated that the MAC regulates cell function by promoting the expression of pro-inflammatory mediators, including adhesion molecules and pro-inflammatory cytokines. We examined, using C6-deficient and C6-sufficient rabbits, the regulatory role of the MAC in mediating IL-8 expression and subsequent neutrophil recruitment in the setting of myocardial ischemia/reperfusion injury. C6-deficient and C6-sufficient rabbits were subjected to 30 min of regional myocardial ischemia followed by a period of reperfusion. In addition to a significant reduction in myocardial infarct size in C6-deficient animals, analysis of myocardial tissue demonstrated a decrease in neutrophil influx into the infarcted region. The reduction in neutrophil influx correlated with the decreased expression of the neutrophil chemotactic cytokine IL-8, as determined by ELISA and immunohistochemical analysis. The results derived from this study provide evidence that the MAC has an important function in mediating the recruitment of neutrophils to the reperfused myocardium through the local induction of IL-8.