Modulation of DNA base excision repair (BER) has the potential to enhance response to chemotherapy and improve outcomes in tumours such as melanoma and glioma. APE1, a critical protein in BER that processes potentially cytotoxic abasic sites (AP sites), is a promising new target in cancer. In the current study, we aimed to develop small molecule inhibitors of APE1 for cancer therapy.
An industry-standard high throughput virtual screening strategy was adopted. The Sybyl8.0 (Tripos, St Louis, MO, USA) molecular modelling software suite was used to build inhibitor templates. Similarity searching strategies were then applied using ROCS 2.3 (Open Eye Scientific, Santa Fe, NM, USA) to extract pharmacophorically related subsets of compounds from a chemically diverse database of 2.6 million compounds. The compounds in these subsets were subjected to docking against the active site of the APE1 model, using the genetic algorithm-based programme GOLD2.7 (CCDC, Cambridge, UK). Predicted ligand poses were ranked on the basis of several scoring functions. The top virtual hits with promising pharmaceutical properties underwent detailed in vitro analyses using fluorescence-based APE1 cleavage assays and counter screened using endonuclease IV cleavage assays, fluorescence quenching assays and radiolabelled oligonucleotide assays. Biochemical APE1 inhibitors were then subjected to detailed cytotoxicity analyses.
Several specific APE1 inhibitors were isolated by this approach. The IC50 for APE1 inhibition ranged between 30 n and 50 μ. We demonstrated that APE1 inhibitors lead to accumulation of AP sites in genomic DNA and potentiated the cytotoxicity of alkylating agents in melanoma and glioma cell lines.
Our study provides evidence that APE1 is an emerging drug target and could have therapeutic application in patients with melanoma and glioma.
melanoma; glioma; DNA repair; human apurinic/apyrimidinic endonuclease 1 (APE1); small molecule inhibitors
Conclusions that can be drawn from earlier studies on noise and children's blood pressure are limited due to inconsistent results, methodological problems, and the focus on school noise exposure.
To investigate the effects of aircraft and road traffic noise exposure on children's blood pressure and heart rate.
Participants were 1283 children (age 9–11 years) attending 62 primary schools around two European airports. Data were pooled and analysed using multilevel modelling. Adjustments were made for a range of socioeconomic and lifestyle factors.
After pooling the data, aircraft noise exposure at school was related to a statistically non‐significant increase in blood pressure and heart rate. Aircraft noise exposure at home was related to a statistically significant increase in blood pressure. Aircraft noise exposure during the night at home was positively and significantly associated with blood pressure. The findings differed between the Dutch and British samples. Negative associations were found between road traffic noise exposure and blood pressure, which cannot be explained.
On the basis of this study and previous scientific literature, no unequivocal conclusions can be drawn about the relationship between community noise and children's blood pressure.
aircraft noise; blood pressure; road traffic noise; heart rate
We conducted a single-centre, randomised, double-blinded, placebo-controlled phase II clinical study to test safety and efficacy of a 12-week therapy with low-dose (700 mg/daily) or high-dose (2800 mg/daily) of NAC.
Twenty-one patients (ΔF508 homo/heterozygous, FEV1 > 40% pred.) were included in the study. After a 3-weeks placebo run-in phase, 11 patients received low-dose NAC, and 10 patients received high-dose NAC. Outcomes included safety and clinical parameters, inflammatory (total leukocyte numbers, cell differentials, TNF-α, IL-8) measures in induced sputum, and concentrations of extracellular glutathione in induced sputum and blood.
High-dose NAC was a well-tolerated and safe medication. High-dose NAC did not alter clinical or inflammatory parameters. However, extracellular glutathione in induced sputum tended to increase on high-dose NAC.
High-dose NAC is a well-tolerated and safe medication for a prolonged therapy of patients with CF with a potential to increase extracellular glutathione in CF airways.
Cystic fibrosis; N-acetylcysteine; induced sputum; glutathione; inflammation
Neutron radiation offers significant advantages for the study of biological molecular structure and dynamics. A broad and significant effort towards instrumental and methodological development to facilitate biology experiments at neutron sources worldwide is reviewed.
Neutron scattering; Neutron crystallography; Small angle neutron scattering; Reflectometry; Inelastic neutron scattering; Quasi-elastic neutron scattering; Proteins; Membranes; Macromolecular structure and dynamics
Background: In epidemiological studies of the potential health effects of traffic related air pollution, self reported traffic intensity is a commonly used, but rarely validated, exposure variable.
Methods: As part of a study on the impact of Traffic Related Air Pollution on Childhood Asthma (TRAPCA), data from 2633 and 673 infants from the Dutch and the German-Munich cohorts, respectively, were available. Parents subjectively assessed traffic intensity at the home address. Objective exposures were estimated by a combination of spatial air pollution measurements and geographic information system (GIS) based modelling using an identical method for both cohorts.
Results: The agreement rates between self reported and GIS modelled exposure—accumulated over the three strata of self assessed traffic intensity—were 55–58% for PM2.5, filter absorbance (PM2.5 abs), and nitrogen dioxide in Munich and 39–40% in the Netherlands. Of the self reported low traffic exposed group, 71–73% in Munich and 45–47% in the Netherlands had low modelled exposure to these three air pollutants. Of the self assessed high exposed subgroups in Munich (15% of the total population) and the Netherlands (22% of the total population), only 22–33% and 30–32% respectively had high modelled exposure to the three air pollutants. The subjective assessments tend to overestimate the modelled estimates for PM2.5 and NO2 in both study areas. When analysis was restricted to the portion of the Dutch cohort living in non-urban areas, the agreement rates were even lower.
Conclusions: Self reported and modelled assessment of exposure to air pollutants are only weakly associated.
Background: Up to 70% of young Nigerian children have been reported to have blood lead concentrations ⩾10 µg/dl.
Aims: To better elucidate risk factors for lead toxicity among Nigerian families with children at risk for lead toxicity.
Methods: Two geographic wards in Jos, Nigeria were selected for study, one previously reported to have a high mean blood lead level (37 (SD 13) µg/dl) and one with a lower mean blood lead level (17 (SD 10) µg/dl) in young children. Data pertaining to potential risk factors for lead exposure were collected from children and adults in 34 households.
Results: The mean (SD) blood lead concentration of 275 subjects, aged 3 weeks to 90 years, was 8.7 (5.7) µg/dl (range 1–34 µg/dl); 92 (34%) had concentrations ⩾10 µg/dl. In multivariate analysis, an age of 5 years and under, flaking house paint, residence near a gasoline seller, male gender, increasing maternal and paternal education, and use of a lead ore eye cosmetic were independently associated with greater blood lead concentration. Vehicle ownership was associated with reduced lead concentration. Compared with the low-lead ward, residence in the high-lead ward remained significantly associated with greater lead values, indicating that additional factors likely contribute to lead exposure.
Conclusion: Although the cause of increased lead levels in Jos appears to be multi-factorial, several remediable sources contribute to lead exposure in Nigeria.
Aims: To determine the response to oral calcium in Nigerian children with rickets.
Methods: In a teaching hospital in Western Nigeria, 26 children (13 boys, 13 girls, aged 2–5 years) with confirmed rickets received calcium lactate (2.7 g/day).
Results: Within one month of treatment leg pain was relieved and the children were more active. The mean x ray score improved from 3.3 at baseline to 1.7 at three months and 0.9 at six months (arbitrary scoring system, 0–6). Twelve cases were healed radiologically after six months, 11 others improved considerably, two showed no significant improvement, and a non-compliant patient was worse. There was progressive reversal of biochemical features. Median plasma alkaline phosphatase fell from 519 (range 178–1078) to 283 (209–443) IU/l (p = 0.04) in four months, while mean 1,25-dihydroxyvitamin D fell from 473 (251–1057) to 281 (155–481) pmol/l (p = 0.04), and mean plasma calcium increased from 2.26 (1.63–2.54) to 2.37 (2.06–2.54) mmol/l (p = 0.13). Parathyroid hormone fell from 5.3 (0.4–21.5) to 1.7 (0.45–7.4) pmol/l. Type I collagen carboxy terminal cross linked telopeptide was very high at baseline (20 (7.2–103) to 14 (11–24) µg/l) (p = 0.03) and fell promptly to normal.
Conclusion: Calcium supplementation alone effected healing of rickets in most of these Nigerian children and may provide sufficient treatment in this environment.
Methods: Levels of eNO in a sample of 450 children aged 7–12 years out of a total sample of 2504 school children living in different urban areas near motorways were determined. The aim of this cross-sectional study was to explore the relationship between eNO, impairment of lung function (PEF, FVC, FEV1 and MMEF), bronchial hyperresponsiveness (BHR), and blood eosinophilia in children with and without atopy as assessed by skin prick testing.
Results: Regression analysis showed that wheezing and nasal discharge and conjunctivitis that had occurred during the previous 12 months were positively associated with eNO levels in atopic children (relative increase of 1.48 and 1.41, respectively; p<0.05) but not in non-atopic children. Similarly, BHR and the number of blood eosinophils per ml were positively associated with eNO levels in atopic children (relative increase of 1.55 and 2.29, respectively; p<0.05) but not in non-atopic children. The lung function indices PEF, FVC, FEV1 and MMEF were not associated with eNO levels.
Conclusions: In addition to conventional lung function tests and symptom questionnaires, eNO is a suitable measure of airway inflammation and its application may reinforce the power of epidemiological surveys on respiratory health.
The major American tobacco companies developed and agreed to abide by the Cigarette Advertising Code in 1964. The stated aims of the code were to prohibit advertising directed at young people, to prohibit advertising that used fraudulent health claims, and to assure compliance with the code's provisions through the establishment of an administrative arm and enforcement mechanism to prescreen and monitor all cigarette advertising. In the 32 years since the Cigarette Advertising Code's adoption, the tobacco industry has used the existence of this code and its revisions and promises of self- regulation in accordance with this code as evidence that it promotes tobacco use only in a responsible manner. The code has served as the basis of the industry's efforts to avoid further local, state, and federal regulatory oversight of its marketing activities. A historical review of cigarette advertising since 1964 indicates that the voluntary code's major provisions have been regularly violated in the spirit and the letter. The administrative and enforcement provisions of the original Cigarette Advertising Code were quietly dismantled soon after the voluntary code's adoption and were completely omitted from the revised code in 1990. The historical evidence indicates that self- regulation of cigarette advertising and promotion by the tobacco industry has been repeatedly given trials and has not worked.
OBJECTIVE: To examine the relation between birth weight and cognitive function in young adult life. DESIGN: Retrospective cohort study based on birth registry data and cognitive function measured during evaluation for military service. SUBJECTS: 4300 Danish conscripts born between 1973 and 1975. MAIN OUTCOME MEASURES: Mean score in the Boerge Prien test of cognitive function; score is the number of correct answers to 78 questions and correlates with full scale intelligence quotient (IQ). RESULTS: Mean score in the Boerge Prien test increased from 39.9 at a birth weight of < or = 2500 g to 44.6 at a birth weight of 4200 g even after adjustment for gestational age and length at birth, maternal age and parity, and other variables. Above a birth weight of 4200 g the test score decreased slightly. CONCLUSION: Birth weight is associated with cognitive performance in young adult life. Interference with fetal growth may influence adult cognitive performance.
Ralstonia eutropha JMP 134 utilizes 3-nitrophenol as the sole source of nitrogen, carbon, and energy. The entire catabolic pathway of 3-nitrophenol is chromosomally encoded. An initial NADPH-dependent reduction of 3-nitrophenol was found in cell extracts of strain JMP 134. By use of a partially purified 3-nitrophenol nitroreductase from 3-nitrophenol-grown cells, 3-hydroxylaminophenol was identified as the initial reduction product. Resting cells of R. eutropha JMP 134 metabolized 3-nitrophenol to N-acetylaminohydroquinone under anaerobic conditions. With cell extracts, 3-hydroxylaminophenol was converted into aminohydroquinone. This enzyme-mediated transformation corresponds to the acid-catalyzed Bamberger rearrangement. Enzymatic conversion of the analogous hydroxylaminobenzene yields a mixture of 2- and 4-aminophenol.
The alpha-glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) is an inhibitor of human immunodeficiency virus (HIV) replication and HIV-induced syncytium formation in vitro. Although an NB-DNJ-mediated change in viral envelope N-glycan composition inhibits HIV entry at the level of post-CD4 binding, the exact mechanism of inhibition remains to be established. In this study we have examined the effects of NB-DNJ on virion envelope composition and CD4-induced gp120 shedding and gp41 exposure. Virion composition analysis revealed an NB-DNJ-mediated reduction of 15% in overall virion envelope glycoprotein content and a reduction of 26% in the proteolytic maturation of virion gp160. Taken together, these two effects resulted in a reduction of approximately 40% in virion gp120 content. CD4-induced shedding of gp120 from the surfaces of envelope-transfected Cos cells was undetectable when gp120 was expressed in the presence of NB-DNJ. Similarly, the shedding of virion-associated gp120 was reduced 7.4-fold. CD4-induced exposure of cryptic gp41 epitopes on the surfaces of HIV-expressing ACH-2 cells was also greatly impaired, and the exposure of virion-associated gp41 epitopes was reduced 4.0-fold. Finally, CD4-induced increases in the binding of antibodies to the V3 loop of ACH-2-cell-expressed envelope glycoproteins were reduced 25-fold when the glycoproteins were expressed in the presence of NB-DNJ. These results suggest that the NB-DNJ-mediated retention of glycosylated N-glycans inhibits HIV entry by a combined effect of a reduction in virion gp120 content and a qualitative defect within the remaining gp120, preventing it from undergoing conformational changes after CD4 binding.
The alpha-glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) is an inhibitor of human immunodeficiency virus (HIV) replication and HIV-induced syncytium formation in vitro. Although NB-DNJ appears to inhibit HIV entry at the level of post-CD4 binding (P.B. Fischer, M. Collin, G.B. Karlsson, W. James, T.D. Butters, S.J. Davis, S. Gordon, R.A. Dwek, and F.M. Platt, J. Virol. 69:5791-5797, 1995), the exact mechanism of action remains to be established. In this study we have examined the effect of NB-DNJ on the structure of recombinant gp120 (rgpl20), expressed in CHO cells, by using a panel of 40 monoclonal antibodies. The levels of binding of antibodies to rgp120 produced in the presence [rgpl20(+)] and absence [rgpl20(-)] of NB-DNJ were compared by enzyme-linked immunosorbent assay and surface plasmon resonance (BIAcore; Pharmacia). The results showed an increase in the binding to rgp120(+) of antibodies directed against the C1 and C2 regions and a decrease in the binding of antibodies directed against the V1/V2 loops compared with antibody binding to rgpl20(-). A decrease in the binding to rgpl20(+) of antibodies directed against discontinuous epitopes was also observed. No differences were seen in the binding of antibodies directed against the crown of the V3 loop and the C4 region of gp120. Treatment of rgpl20 with alpha-glucosidases I and II had no effect on the differential binding observed, whereas treatment with sialidase abolished the differences seen in the binding of antibodies directed against the C1 and C2 regions of gp120. In addition to these findings, rgpl20(+) showed increased sensitivity to proteases released by CHO cells during expression, as well as to exogenous thrombin. Taken together, the data presented in this paper suggest that production of gp120 in the presence of NB-DNJ affects the conformation of the Vl/V2 loops of gpl20, as well as the overall charge of the C1 and C2 regions. These effects may play a role in the previously described NB-DNJ-mediated inhibition of HIV entry at the level of post-CD4 binding.
The alpha-glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) is a potent inhibitor of human immunodeficiency virus (HIV) replication and syncytium formation in vitro. However, the exact mechanism of action of NB-DNJ remains to be determined. In this study we have examined the impairment of HIV infectivity mediated by NB-DNJ. By two independent HIV entry assays [PCR-based HIV entry assay and entry of Cocal(HIV) pseudotypes], the reduction in infectivity was found to be due to an impairment of viral entry. No effect of NB-DNJ treatment was seen on the kinetics of the interaction between gp120 and CD4 (surface plasmon resonance; BIAcore) or on the binding of virus particles to H9 cells (using radiolabeled virions). We therefore conclude that a major mechanism of action of NB-DNJ as an inhibitor of HIV replication is the impairment of viral entry at the level of post-CD4 binding, due to an effect on viral envelope components.
The conversion of 2-chloro-cis,cis-muconate by muconate cycloisomerase from Pseudomonas putida PRS2000 yielded two products which by nuclear magnetic resonance spectroscopy were identified as 2-chloro- and 5-chloromuconolactone. High-pressure liquid chromatography analyses showed the same compounds to be formed also by muconate cycloisomerases from Acinetobacter calcoaceticus ADP1 and Pseudomonas sp. strain B13. During 2-chloro-cis,cis-muconate turnover by the enzyme from P. putida, 2-chloromuconolactone initially was the major product. After prolonged incubation, however, 5-chloromuconolactone dominated in the resulting equilibrium. In contrast to previous assumptions, both chloromuconolactones were found to be stable at physiological pH. Since the chloromuconate cycloisomerases of Pseudomonas sp. strain B13 and Alcaligenes eutrophus JMP134 have been shown previously to produce the trans-dienelactone (trans-4-carboxymethylene-but-2-en-4-olide) from 2-chloro-cis,cis-muconate, they must have evolved the capability to cleave the carbon-chlorine bond during their divergence from normal muconate cycloisomerases.
A bacterial strain, Mycobacterium sp. strain HL 4-NT-1, enriched with 4-nitrotoluene as its sole source of nitrogen, was able to metabolize 2,4,6-trinitrotoluene under aerobic conditions. The dark red-brown metabolite, which accumulated in the culture fluid, was identified as a hydride-Meisenheimer complex by comparison with an authentic synthetic sample.
Angular dioxygenation has been established as the crucial step in dibenzofuran degradation by Brevibacterium sp. strain DPO 1361 (V. Strubel, K. H. Engesser, P. Fischer, and H.-J. Knackmuss, J. Bacteriol. 173:1932-1937, 1991). The same strain utilizes biphenyl and fluorene as sole sources of carbon and energy. The fluorene degradation sequence is proposed to be initiated by oxidation of the fluorene methylene group to 9-fluorenol. Cells grown on fluorene exhibit pronounced 9-fluorenol dehydrogenase activity. Angular dioxygenation of the 9-fluorenone thus formed yields 1,10-dihydro-1,10-dihydroxyfluoren-9-one (DDF). A mechanistic model is presented for the subsequent C-C bond cleavage by an NAD(+)-dependent DDF dehydrogenase, acting on the angular dihydrodiol. This enzyme was purified and characterized as a tetramer of four identical 40-kDa subunits. The following Km values were determined: 13 microM for DDF and 65 microM for 2,3-dihydro-2,3-dihydroxybiphenyl. The enzyme also catalyzes the production of 3-(2'-carboxyphenyl)catechol, which was isolated, and structurally characterized, in the form of the corresponding lactone, 4-hydroxydibenzo-(b,d)-pyran-6-one. Stoichiometry analysis unequivocally demonstrates that angular dioxygenation constitutes the principal pathway in Brevibacterium sp. strain DPO 1361.
The sensitivity and specificity of Hachinski's Ischaemic Score (IS) in the diagnosis of the vascular aetiology of dementia was studied in a series of 32 demented patients, dementia of the Alzheimer type (16), multi-infarct dementia (7), mixed dementia (6), Pick's disease (3), with neuropathological diagnosis as the point of reference. The IS distinguished between primary degenerative dementia and multi-infarct or mixed dementia. As single features of the IS "a positive history of stroke" and "a fluctuating course" showed differing prevalences in the latter two diagnostic categories. The IS labelled 21% of patients with primary degenerative dementia as having a vascular aetiology. The uncritical application of the IS to large samples in epidemiological studies may cause incorrect labelling of a significant proportion of patients with primary degenerative dementia as vascular dementia. These results are based on observations of long-term inpatients and depend on neuropathological criteria. While the definite diagnosis of DAT by threshold criteria concerning plaque and tangle counts is well established, neither clinical nor pathological evidence of stroke necessarily means that cerebrovascular disease has anything to do with a patient's dementia.
Brevibacterium sp. strain DPO 1361 oxygenates dibenzofuran in the unusual angular position. The 3-(2-hydroxyphenyl)catechol thus generated is subject to meta ring cleavage in the proximal position, yielding 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid, which is hydrolyzed to 2-oxo-4-pentenoate and salicylate by 2-hydroxy-6-oxo-6-phenyl-2,4-hexadienoic acid hydrolase. The proximal mode of ring cleavage is definitely established by isolation and unequivocal structural characterization of a cyclization product of 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid, i.e., 3-(chroman-4-on-2-yl)pyruvate.
Enzymatic conversion of 4-fluorocatechol in the simultaneous presence of partially purified preparations of catechol 1,2-dioxygenase from Pseudomonas cepacia and muconate cycloisomerase from Alcaligenes eutrophus 335 yielded a product that was unambiguously identified as (+)-4-fluoromuconolactone [(+)-4-carboxymethyl-4-fluoro-but-2-en-4-olide]. This compound was shown to be the only major product formed from 3-fluoro-cis,cis-muconate by the action of muconate cycloisomerases from A. eutrophus 335, A. eutrophus JMP134, and P. cepacia as well as by the action of dichloromuconate cycloisomerase from A. eutrophus JMP134. This finding implies that dichloromuconate cycloisomerase, like the muconate cycloisomerases, catalyzes primarily a cycloisomerization reaction, which only in the case of chloro- and bromo-substituted substrates is connected to a dehalogenation. 4-Fluoromuconolactone at pH 7 decomposes by spontaneous reactions mainly to maleylacetate, which then decarboxylates to give cis-acetylacrylate. Although significant amounts of an unidentified compound are also formed from the fluorolactone, HF elimination to the two isomeric dienelactones (4-carboxymethylenebut-2-en-4-olides) is negligible. However, all spontaneous reactions proceed so slowly that an enzymatic conversion of 4-fluoromuconolactone must be assumed. Participation of dienelactone hydrolases in this reaction is indicated by their induction during growth of various strains with 4-fluorobenzoate. However, experiments with cell extracts of P. putida A3.12 suggest that at least one other hydrolytic enzyme is able to contribute to 4-fluoromuconolactone conversion. In light of these observations, earlier proposals for a 4-fluorobenzoate degradative pathway are discussed.