Effect of sequence context on O(6)-methylguanine repair and replication in vivo.

Understanding the origins of mutational hotspots is complicated by the intertwining of several variables. The selective formation, repair, and replication of a DNA lesion, such as O(6)-methylguanine (m(6)G), can, in principle, be influenced by the surrounding nucleotide environment. A nearest-neighbor analysis was used to address the contribution of sequence context on m(6)G repair by the Escherichia coli methyltransferases Ada or Ogt, and on DNA polymerase infidelity in vivo. Sixteen M13 viral genomes with m(6)G flanked by all permutations of G, A, T, and C were constructed and individually transformed into repair-deficient and repair-proficient isogenic cell strains. The 16 genomes were introduced in duplicate into 5 different cellular backgrounds for a total of 160 independent experiments, for which mutations were scored using a recently developed assay. The Ada methyltransferase demonstrated strong 5' and 3' sequence-specific repair of m(6)G in vivo. The Ada 5' preference decreased in the general order: GXN > CXN > TXN > AXN (X = m(6)G, N = any base), while the Ada 3' preference decreased in the order: NX(T/C) > NX(G/A), with mutation frequencies (MFs) ranging from 35% to 90%. The Ogt methyltransferase provided MFs ranging from 10% to 25%. As was demonstrated by Ada, the Ogt methyltransferase repaired m(6)G poorly in an AXN context. When both methyltransferases were removed, the MF was nearly 100% for all sequence contexts, consistent with the view that the replicative DNA polymerase places T opposite m(6)G during replication irrespective of the local sequence environment.

Simultaneous bilateral spontaneous pneumothoraces in a young woman with anorexia nervosa.

UNLABELLED: An eighteen year-old woman with anorexia nervosa was admitted to hospital with acute dyspnea. There was no history of trauma to the chest and the patient denied any vomiting. METHOD: A chest radiograph was taken which showed bilateral pneumothoraces with complete collapse of both lungs. RESULTS: Following insertion of bilateral chest drains the patient made a complete recovery, later having bilateral pleurectomies performed to prevent recurrence. DISCUSSION: We discuss the evidence suggesting that the state of malnutrition that results from anorexia nervosa may in fact predispose patients to the development of pneumothoraces.

The development of a psychodynamic treatment for patients with borderline personality disorder: a preliminary study of behavioral change.

This study examines the effectiveness of a modified psychodynamic treatment called Transference Focused Psychotherapy (TFP) designed specifically for patients, with borderline personality disorder (BPD). Twenty-three female patients diagnosed with DSM-IV BPD began twice-weekly TFP. Patients were assessed at baseline and at the end of 12 months of treatment with diagnostic instruments, measures of suicidality, self-injurious behavior, and measures of medical and psychiatric service utilization. Compared to the year prior to treatment, the number of patients who made suicide attempts significantly decreased, as did the medical risk and severity of medical condition following self-injurious behavior. Compared to the year prior, study patients during the treatment year had significantly fewer hospitalizations as well as number and days of psychiatric hospitalization. The dropout rate was 19.1%. This uncontrolled study is highly suggestive that this structured and manualized psychodynamic treatment modified for borderline patients shows promise for the ambulatory treatment of these patients and warrants further study.

Context-dependent mutagenesis by DNA lesions.

BACKGROUND: Detailed analyses of mutational hotspots following DNA damage provide an understanding of oncogene activation and tumor suppressor gene inactivation, and hence provide an insight into the earliest steps in the induction of cancer. A mutational hotspot might be created by preferential lesion formation, decreased lesion repair, or increased misinsertion past the lesion during DNA replication. The respective contribution of these factors might be influenced by the DNA sequence context of the hotspot. RESULTS: As a prelude to addressing the contribution of all possible nearest-neighbor contexts on the replication past O6-methylguanine (m6G) and repair of m6G in vivo, we have devised a mutation frequency (MF) detection strategy on the basis of the properties of type IIs restriction enzymes. We also report a method for constructing site-specific single-stranded viral DNA genomes that should yield identical ligation efficiencies regardless of the lesion or its surrounding sequence context. Using repair-deficient Escherichia coli, we discovered that m6G in three sequence contexts was nearly 100% mutagenic in vivo, showing that the DNA polymerase holoenzyme almost always placed a thymine base opposite m6G during replication. In partially repair-proficient cells, the Ada O6-methylguanine-DNA methyltransferase repair protein was twice as efficient on m6G when a guanine base rather than an adenine base was 5' to the lesion. CONCLUSIONS: The system allows the mutagenic potential of, theoretically, any DNA lesion that exhibits point mutations, in any varied local sequence context, to be rapidly determined. The assay demonstrates low background, high throughput, and does not require phenotypic selection, making it possible to discern the effects of sequence context on the processing of m6G.

Normative reproductive indices for male and female adult Sprague-Dawley rats.

The Sprague-Dawley rat is traditionally used as the experimental model for the study of contraceptive agents and reproductive toxicants. Until recently, the normative values used to compare hormone levels after drug exposure were based on the values generated by radioimmunoassay methods developed 30 years ago. To ascertain normative reproductive indices for adult female and male Sprague-Dawley rats over a 6-month age period, we measured luteinizing hormone, testosterone, and estradiol using commercially available kits that employ updated assay techniques. In addition, sperm indices were correlated with reproductive hormones over the same time period. Animals were killed at 107, 128, 156, 212, and 268 days of age irrespective (for females) of cycle stage. Serum LH levels did not change with increasing age; however, the female rats had significantly higher LH values than did the males at comparable ages (p < 0.001). Testosterone levels and sperm parameters did not significantly change with increasing age. Estradiol levels were significantly higher in 107-day-old female rats than they were in female rats in all older age groups (p < 0.01). The values reported can be used in designing and interpreting data generated in ongoing, long term toxicological and contraceptive studies using the rat animal model.

Mutational analysis of the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor. A consensus casein kinase II site followed by 2 leucines near the carboxyl terminus is important for intracellular targeting of lysosomal enzymes.

The cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (CI-MPR) mediates intracellular sorting of lysosomal enzymes, binding lysosomal enzymes in the Golgi and delivering them to a lysosomal compartment. The receptor also mediates endocytosis of extracellular ligands. We have devised a new method that rigorously measures function of the CI-MPR in intracellular sorting and used it to identify a previously uncharacterized signal near the COOH terminus of the receptor that is required for sorting. We stably transfect mutant receptors into CI-MPR-deficient mouse L cells, isolate homogeneous clonal cell lines that express a range of receptor levels for each mutant, and assay each cell line for levels of receptor expression and secretion of total phosphorylated lysosomal enzymes. Examination of the secretion phenotype of the cells as a function of receptor levels provides a sensitive indicator of the intrinsic sorting efficiency of each mutant receptor. We find that chimeric CI-MPRs that contain the bovine extracytoplasmic domain and the human or mouse transmembrane and cytoplasmic domains function identically to the bovine receptor, thus demonstrating that sorting signals are conserved. Analysis of a series of truncation and alanine scanning mutants reveals that a consensus casein kinase II site followed by 2 leucines near the COOH terminus that has the sequence (-10)DDSDEDLL(-3) is important for receptor function in sorting of lysosomal enzymes.

Endoscopic bougie and balloon dilatation of multiple bronchial stenoses: 10 year follow up.

This paper reports 10 year follow up data on four patients who underwent mechanical dilatation of multiple bronchial stenoses (sarcoidosis two cases, berylliosis one case, idiopathic stenoses one case). Two patients enjoyed symptomatic and physiological improvement during the nine years before they presented with a recurrence of stenosis. The other two had recurrences sooner, and improved only slightly after they had started prednisolone treatment.

Pressurised aerosol inhalers: the cost of misuse.

Bronchodilator aerosols, if used correctly, have many advantages over other therapies in patients with chronic airflow limitation caused by asthma or chronic bronchitis. The use of pressurized aerosol inhalers was examined in a district general hospital: of 57 patients on these inhalers, 39 were unable to use the inhaler effectively, and 23 had never received any advice on inhaler technique. A single demonstration of correct technique decreased the failures to 21 patients and, after two demonstrations, to ten. The cost of the misused inhalers in this relatively small population was 450 pounds, and obviously this figure escalates when the prescription for these inhalers is repeated monthly. It is also increased when the total numbers of misused inhalers in the country are accounted for. The cost in terms of finance, in these days of medical audit and drug budgets, and, more importantly, in terms of patient health, is unacceptable and can be avoided by repeated tuition of technique.

Locally invasive pulmonary aspergillosis occurring in a gardener: an occupational hazard?

Fatal locally invasive pulmonary aspergillosis occurred in a previously fit young patient who had no predisposing factors other than exposure to fungal spores in his occupation as a gardener.

Prolonged bleeding times after aspirin ingestion in patients with aspirin idiosyncrasy.

Measurements of the bleeding time after the ingestion of small doses of aspirin in patients with aspirin idiosyncrasy demonstrate a marked prolongation compared with control patients. Confirmation of this finding offers a clue to the pathogenesis of this condition. Despite the prolonged bleeding time, gastrointestinal haemorrhage is an uncommon event in patients with aspirin idiosyncrasy.

Complement components and IgE in patients with asthma and aspirin idiosyncrasy.

Levels of circulating IgE, total haemolytic complement, and components C4 and C3 were measured in 16 asthmatics with aspirin idiosyncrasy and in control subjects. IgE levels were mostly within normal limits. No differences were found between the complement profiles-in particular the C4 levels-in the two groups. As low levels of C4 have been found in patients with intrinsic asthma, these results would suggest a fundamental difference between asthmatics with aspirin idiosyncrasy and others with intrinsic asthma.

The effect of sodium cromoglycate on analgesic-induced asthmatic reactions.

The onset of increased airways obstruction after analgesic ingestion by asthmatics with analgesic idiosyncrasy suggests an immediate type 1 allergic response. The effect of sodium cromoglycate (DSCG) on this response was assessed in nineteen patients with this syndrome. DSCG prevented asthmatic attacks in only three patients, all non-atopic, suggesting that analgesic idiosyncrasy is not an allergic disorder, The effectiveness of DSCG in preventing asthmatic attacks in some patients with analgesic idiosyncrasy would suggest that a trial of this preparation should be undertaken before commencing continuous corticosteroid therapy.