Induction of O6-alkylguanine-DNA-alkyltransferase in the hepatocytes of rats following treatment with 2-acetylaminofluorene.

Molecular and immunohistological techniques have been used to study the induction in rat liver of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (ATase), following an acute dose (60 mg/kg) of the hepatocarcinogen, 2-acetylaminofluorene (2-AAF). An increase in ATase activity was specific to the liver, with a five- to six-fold induction being observed 72 hr after administration of 2-AAF. A similar temporal increase of both activity and ATase protein (detected by immunoblotting) was observed up to 1 week following treatment, but after 2 weeks the activity had returned to control levels.

Apolipoprotein E genotypes and serum lipid levels in Alzheimer's disease and multi-infarct dementia.

Objective: Assessment of apolipoprotein E genotype, serum cholesterol, triglycerides, high density lipoprotein-cholesterol and low density lipoprotein-cholesterol levels in different types of dementia.

Subjects: 102 consecutive referrals to an old age psychiatry service based at Manchester were classified according to clinical criteria based on ICD 10.

Results: Thirty-seven were considered to have Alzheimer's disease, 16 multi-infarct dementia and 33 to be free from dementia.

O6-benzylguanine potentiates the in vivo toxicity and clastogenicity of temozolomide and BCNU in mouse bone marrow.

The effects of treatment of mice with O6-benzylguanine (O6-BeG) on the levels of O6-alkylguanine-DNA alkyltransferase (ATase) in the hematopoietic compartment and on the in vivo sensitivity of hematopoietic progenitor cells to the toxic and clastogenic effects of the antitumor agents 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) and temozolomide were studied. When the overall effects of BCNU alone or with O6-BeG pretreatment were compared, dose potentiating factors of 4.17 for marrow cellularity, 4.

[Will transfer of cytostatic drug resistance genes increase hematopoiesis resistance in the treatment of malignant tumors?].

The aim of aggressive antitumor chemotherapy is to kill the tumor with the largest possible dose of a cytotoxic drug. The maximum dose tolerated by the patient is limited by the toxicity to normal tissue, hematopoiesis being frequently the most sensitive system. Transfer of drug resistance genes to hematopoietic cells could protect them against chemotherapy-related toxicity and thus could be a way of gene therapy in cancer.

Crystallization of E. coli RuvA gives insights into the symmetry of a Holliday junction/protein complex.

The E. coli protein RuvA (resistance to ultraviolet light) has been overexpressed in E. coli, purified and crystallized using the hanging-drop vapour-diffusion method with sodium chloride as the precipitant.

A mechanism of drug action revealed by structural studies of enoyl reductase.

Enoyl reductase (ENR), an enzyme involved in fatty acid biosynthesis, is the target for antibacterial diazaborines and the front-line antituberculosis drug isoniazid. Analysis of the structures of complexes of Escherichia coli ENR with nicotinamide adenine dinucleotide and either thienodiazaborine or benzodiazaborine revealed the formation of a covalent bond between the 2' hydroxyl of the nicotinamide ribose and a boron atom in the drugs to generate a tight, noncovalently bound bisubstrate analog. This analysis has implications for the structure-based design of inhibitors of ENR, and similarities to other oxidoreductases suggest that mimicking this molecular linkage may have generic applications in other areas of medicinal chemistry.

Crystallization of Escherichia coli enoyl reductase and its complex with diazaborine.

Recent work has shown that the NADH-dependent enoyl acyl carrier protein reductase from Escherichia coli is the target for diazaborine, an antibacterial agent. This enzyme has been crystallized by the hanging-drop method of vapour diffusion complexed with NAD(+) and in the presence and absence of a thieno diazaborine. The crystals grown in the absence of diazaborine (form A) are in the space group P2(1) with unit-cell dimensions a = 74.

Pudendal nerve terminal motor latency influences surgical outcome in treatment of rectal prolapse.

Purpose: This study was undertaken to document the effect of pudendal nerve function on anal incontinence after repair of rectal prolapse.

Methods: Patients with full rectal prolapse (n = 24) were prospectively evaluated by anal manometry and pudendal nerve terminal motor latency (PNTML) before and after surgical correction of rectal prolapse (low anterior resection (LAR; n = 13) and retrorectal sacral fixation (RSF; n = 11)).

Results: Prolapse was corrected in all patients; there were no recurrences during a mean 25-month follow-up.

Crystal structure of DNA recombination protein RuvA and a model for its binding to the Holliday junction.

The Escherichia coli DNA binding protein RuvA acts in concert with the helicase RuvB to drive branch migration of Holliday intermediates during recombination and DNA repair. The atomic structure of RuvA was determined at a resolution of 1.9 angstroms.

Protection of mammalian cells against chloroethylating agent toxicity by an O6-benzylguanine-resistant mutant of human O6-alkylguanine-DNA alkyltransferase.

Low levels of expression in haemopoietic cells of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (A Tase), is associated with the dose-limiting sensitivity of these cells to the chemotherapeutic chloroethylating and related methylating agents. Thus, the use of agents which deplete ATase such as O6-benzylguanine (O6-beG), as a tumour sensitisation strategy is likely further to potentiate collateral toxicity in bone marrow. In order to address this problem, we have engineered two mutants of human ATase (hAT) for resistance to O6-beG and characterised the in vitro properties of the proteins.

Chemoprotection of normal tissues by transfer of drug resistance genes.

The effectiveness of many types of antitumour agent is limited by (i) acute dose limiting cytotoxicity, principally myelosuppression but also lung, liver and gastrointestinal tract toxicity, (ii) the risk of therapy related secondary malignancy and (iii) the inherent or acquired drug-resistance of tumour cells. As the management of the acute toxic effects improve, the more insidious effects, and particularly haematological malignancies, are anticipated to increase. Furthermore, attempts to overcome tumour cell resistance to treatment can lead to increased collateral damage in normal tissues.

Contribution of ogt-encoded alkyltransferase to resistance to chloroethylnitrosoureas in nucleotide excision repair-deficient Escherichia coli.

We investigated the relative contribution of the two Escherichia coli DNA alkyltransferases (ATases) to the increased sensitivity of ATase-deficient bacteria to the mutagenic and lethal effects of chloroethylnitrosoureas (CNU). The ogtencoded protein was the principal determinant in resistance to the mutagenic effects of CNU in E.coli.

Overview of meteorological measurements for aerial spray modeling.

The routine meteorological observations made by the National Weather Service have a spatial resolution on the order of 1,000 km, whereas the resolution needed to conduct or model aerial spray applications is on the order of 1-10 km. Routinely available observations also do not include the detailed information on the turbulence and thermal structure of the boundary layer that is needed to predict the transport, dispersion, and deposition of aerial spray releases. This paper provides an overview of the information needed to develop the meteorological inputs for an aerial spray model such as the FSCBG and discusses the different types of instruments that are available to make the necessary measurements.

Long-term protection of hematopoiesis against the cytotoxic effects of multiple doses of nitrosourea by retrovirus-mediated expression of human O6-alkylguanine-DNA-alkyltransferase.

A human O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA-containing retrovirus was used to infect murine long-term primary bone marrow cultures. High levels of ATase expression were obtained, and colony-forming cells of the granulocyte-macrophage lineage from the cultures transduced with the human ATase retrovirus were three times more resistant to the alkylating agent, N-methyl-N-nitrosourea (MNU), than control cultures. Furthermore, expression of the human ATase protected long-term hematopoiesis, measured as the output of progenitor cells to the nonadherent fraction of the culture, against the cytotoxic effects of repeated exposures to MNU.

Induction of murine O6-alkylguanine-DNA-alkyltransferase in response to ionising radiation is p53 gene dose dependent.

Expression of both the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (ATase) and the p53 tumour suppressor protein are inducible by a number of DNA damaging agents. It is probable that DNA strand breaks are the common inducing signals. This similarity, and the function of p53 as a transcription factor lead us to reason that p53 might be involved in ATase inducibility.

Common themes in redox chemistry emerge from the X-ray structure of oilseed rape (Brassica napus) enoyl acyl carrier protein reductase.

Background: Enoyl acyl carrier protein reductase (ENR) catalyzes the NAD(P)H-dependent reduction of trans-delta 2-enoyl acyl carrier protein, an essential step in de novo fatty acid biosynthesis. Plants contain both NADH-dependent and separate NADPH-dependent ENR enzymes which form part of the dissociable type II fatty acid synthetase. Highly elevated levels of the NADH-dependent enzyme are found during lipid deposition in maturing seeds of oilseed rape (Brassica napus).

O6-benzylguanine increases the sensitivity of human primary bone marrow cells to the cytotoxic effects of temozolomide.

The sensitivity of human primary bone marrow granulocyte/macrophage precursor cells to the cytotoxic effects of the methylating antitumor agent temozolomide (8-carbamoyl-3- methylimidazo[5,1-d]-1,2,3,5-tetrazin-4-[3H]-1) was investigated using an in vitro colony-forming assay. In the eight samples examined, there was a range of sensitivities with D37 values from 18.2 to > 55 microM.

Evidence for the simultaneous expression of alternatively spliced alkylpurine N-glycosylase transcripts in human tissues and cells.

We have isolated a novel human alkylpurine N-glycosylase (APNG) cDNA from a placental library by screening with an oligonucleotide based on the published sequence of the human liver cDNA encoding this protein. The nucleotide sequences of the two cDNAs were essentially identical, but the 5' untranslated region of the new sequence was truncated and the 5'-terminal 92 nucleotides of the novel cDNA were different, indicating the possibility of alternative transcripts. This region included a portion of the open reading frame, so that the predicted protein was truncated and the seven N-terminal amino acids differed from the published sequence for APNG.

Role of nucleotide excision repair in processing of O4-alkylthymines in human cells.

O4-Alkylthymines have been implicated as potential carcinogenic DNA lesions. We have studied the effects of O4-methylthymine, O4-ethylthymine, and O4-n-propylthymine in a model system in which a single lesion was located at a defined position on a SV40-based shuttle vector and have found large differences in the effects of these lesions in repair-proficient and nucleotide excision repair-deficient cells. In repair-competent human HeLa cells, normal fibroblasts, and XP-A (2OS) revertant cells, all 3 residues were highly mutagenic; a mutation frequency of approximately 20% was found for both O4-methylthymine and O4-ethylthymine, whereas that of O4-n-propylthymine was approximately 12%.

Burnout and career-choice regret among family practice physicians in early practice.

Objective: Burnout and career-choice regret among physicians in early practice may contribute to physician impairment and attrition as well as patient dissatisfaction.

Method: Fifty residency-trained family physicians in early practice completed the Maslach Burnout Inventory and a questionnaire on career-choice, demographics, and practice characteristics.

Results: The sample showed moderate burnout related to emotional exhaustion and depersonalization of patients but low burnout related to lack of personal accomplishment.

bcl-2 delay of alkylating agent-induced apoptotic death in a murine hemopoietic stem cell line.

Many cytotoxic agents kill cells by invoking a specific death pathway termed physiological cell death, or apoptosis. Treatment of a murine hemopoietic stem cell line, FDCP-mix, with methylmethanesulfonate (MMS) or N'-methyl-N'-nitrosourea (MNU) leads to death by apoptosis. Retroviral gene transfer was used to overexpress the bcl-2 oncogene in FDCP-mix cells, and this was associated with a delay in apoptosis in these cells after treatment with MNU and MMS and decreased sensitivity of colony formation to the cytotoxic effects of MMS.

Detrimental effect of nitric oxide synthase inhibition during endotoxemia may be caused by high levels of tumor necrosis factor and interleukin-6.

Background: Nitric oxide (NO) production increases during sepsis and endotoxemia. Inhibition of NO synthase has been suggested as a therapeutic modality in sepsis and endotoxemia, but in recent reports NO synthase inhibition increased mortality rate. The mechanism of this phenomenon is not known.

The Met repressor-operator complex: DNA recognition by beta-strands.

The crystal structure of the E. coli met repressor in complex with a synthetic 19-base pair oligonucleotide reveals two dimeric repressor molecules bound to adjacent sites on the DNA. The oligonucleotide contains two adjacent repeats of an 8-mer known as a met-box, which represents the consensus of the met operator sites.