'Codeine is my companion': misuse and dependence on codeine containing medicines in Ireland.

OBJECTIVES: Global concern around over the counter availability of codeine containing products and risk of misuse, dependence and related harms are evident. A phenomenological study of lived experiences of codeine misuse and dependence was undertaken in Ireland, following the Pharmaceutical Society of Ireland's 2010 guidelines for restricted supply of non-prescription codeine containing products. METHODS: In-depth interviews were conducted with a purposive sample of adult codeine misusers and dependents (n=21), both actively using, in treatment and in recovery. The narratives were analysed using the Empirical Phenomenological Psychological five-step method (Karlsson, 1995). A total of 10 themes with 82 categories were identified. Two concepts at a higher level of abstraction above the theme-level emerged during the final stage of analysis. The concepts identified were 'emotional pain and user self-legitimization of use' and 'entrapment into habit-forming and invisible dependent use'. These concepts were reported in different ways by a majority of participants. RESULTS: Findings are presented under the following themes: (1) profile and product preferences; (2) awareness of habit forming use and harm; (3) negotiating pharmacy sales; (4) alternative sourcing routes; (5) the codeine feeling; (6) the daily routine; (7) acute and chronic side effects; (8) social isolation; (9) withdrawal and dependence and (10) help-seeking and treatment experiences. CONCLUSIONS: There is a public health and regulatory imperative to develop proactive responses tackling public availability of codeine containing medicines, risk minimisation in consumer self-treatment for pain, enhanced patient awareness of potential for habit forming use and its consequences and continued health professional pharmacovigilence.

The K(m) for Radiosensitization of Human Tumor Cells by Oxygen is Much Greater than 3 mmHg and is Further Increased by Elevated Levels of Cysteine.

We studied the role of cysteine as an intracellular radiation protector under conditions in which both oxygen and thiols were monitored at 37 degrees C. In HCT-116 human colon cancer cells, the intracellular cysteine content affects the radiation survival dramatically at intermediate oxygen levels, but not at zero or high oxygen levels. Using a spin-through-oil method with a dual radioactive label detection system, we measured intracellular cysteine and glutathione (GSH) levels for cells in suspension culture. A comparison of the cysteine levels of monolayer cells lysed in situ and of trypsinized monolayer cells in suspension (Horan et al., Cytometry 29, 76-82, 1997) revealed that, upon trypsinization from monolayer culture and transfer to a spinner apparatus at 37 degrees C, HCT-116 cells lose most of their intracellular cysteine. Over the 60-min time course of control experiments, these cells do not recover intracellular cysteine despite the availability of cystine (the disulfide of cysteine) in the medium. When cells in spinner culture are provided with exogenous cysteine, they initially concentrate it to 10-fold the extracellular concentration, with the concentration factor decreasing to about 5-fold over the course of an hour. The intracellular GSH concentration changes little throughout this period, regardless of the changes in cysteine levels. The same apparatus was used to assess the survival of HCT-116 cells irradiated at 37 degrees C under conditions of constant pO(2) monitoring. For cells without added cysteine, the oxygen concentration for half-maximal radiation sensitivity was about 7.5 mmHg (intermediate hypoxia), more than twice the commonly accepted value (3 mmHg). At 7.5 mmHg, cells with added cysteine (intracellular concentration 3.5 mM) were almost as radioresistant as severely hypoxic cells (approximately 0.005% oxygen). Cells in parallel experiments in which the cells were grown in monolayers on glass Petri dishes had intermediate cysteine values and also intermediate radiosensitivity. We conclude that the radiation response of cells at intermediate oxygen levels is controlled predominantly by intracellular cysteine levels and that the cysteine levels commonly found in tumors may increase the K(m) for radiosensitivity to values much higher than suggested previously.

In vitro effects of photodynamic therapy on Aspergillus fumigatus.

Photodynamic therapy is a technique for killing cells with visible light after pretreatment with a photosensitizing agent. We demonstrated significant in vitro fungicidal activity against Aspergillus fumigatus of the photosensitizer Green 2W, activated with 630 nm light. This effect was both inoculum- and light dose-dependent. At a Green 2W concentration of 31.5 mg/L, there was complete killing of 2.7 x 10(1) cfu/mL with a light dose of 110 J/cm(2) and up to 2.7 x 10(6) cfu/mL with a light dose of 385 J/cm(2).

Combined Doppler, blood gas, and Bennet quick test for venous leak practical for prepenile prosthesis testing in solo private offices.

Cavernosal blood gases have been widely reported but have not become routine in private practice. The method reported here employs the aspiration of blood gases 15 minutes after pharmacologic challenge followed by a quick test for venous leak followed by Doppler stethoscope observations. The results in 90 patients suggest its selective use in the subset of nonresponding, nonleakers who have open cavernous arteries by Doppler, typically diabetics.

Characterization of the biosynthetic gene cluster for the oligosaccharide antibiotic, Evernimicin, in Micromonospora carbonacea var. africana ATCC39149.

Evernimicin (EV) belongs to the orthosomycin class of antibiotics and consists of several modified L- and D-deoxysugars containing unusual orthoester and glycosyl linkages and two orsellinic acid groups, one that is halogenated. The EV biosynthetic gene cluster from Micromonospora carbonacea var. africana ATCC39149 was localized by hybridization to a dTDP-D-glucose 4,6-dehydratase probe and a 120-kb region containing the EV biosynthetic cluster and surrounding regions has been sequenced. BLAST analysis has identified a type I polyketide synthase for orsellinic acid biosynthesis as well as enzymes required for L- and D-deoxyglucose and D-deoxymannose synthesis. In addition, genes involved in glycosyltransfer and resistance were identified. Insertional mutations in several biosynthetic genes blocked EV production, indicating a role for these genes in EV biosynthesis.

Hypoxia and VEGF mRNA expression in human tumors.

High expression of circulating plasma vascular endothelial growth factor (VEGF) in patients with cancer is an indicator of poor treatment response. Similarly, hypoxia in tumors, as measured by oxygen needle electrodes, has been found to predict for tumor-treatment failure. These two predictors may be related because hypoxia is a potent stimulator of VEGF expression in vitro. However, the demonstration of a relationship between hypoxia and VEGF in human tumors has, to date, been indirect or even negative. The purpose of this study was to test whether this unexpected result was caused by factors unique to human tumors, or whether the prior results could have been influenced by the known complexities of VEGF regulation. Therefore, we undertook a direct assessment of VEGF induction in human tumors using in situ hybridization and compared its distribution with that of hypoxia, as measured by the distribution of adducts of the hypoxia marker EF5. The distribution of both markers was assessed in relationship to the distribution of blood vessels, as measured by antibodies to CD31. Our hypothesis was that VEGF mRNA and hypoxia would colocalize, assuming that detectability of the former was not limiting. Four squamous cell carcinomas, three sarcomas and one glioblastoma multiforme were studied. When VEGF mRNA signal was detectable, its maxima colocalized with regional maxima of EF5 binding. The strongest levels of both signals were sometimes adjacent to regions of tissue necrosis. However, we were unable to predict absolute levels of EF5 binding based on absolute levels of VEGF mRNA. Conversely, for all tumors studied, regions with relatively low levels of EF5 binding had relatively low or undetectable VEGF mRNA. We found moderate EF5 binding in some keratinized cells but VEGF mRNA was not expressed by these differentiated cells. The paradigm that hypoxia and VEGF expression are linked in human tumors is supported by the data presented herein. A better understanding of the biology behind VEGF expression, including its modulation by hypoxia, is important for optimizing its use as a prognostic indicator and/or modulating its presence with biologic therapies.

Mean time to cancer-specific death of apparently clinically localized prostate cancer: policy implications for threshold ages in prostate-specific antigen screening and ablative therapy.

OBJECTIVE: To assess the mean time to cancer-specific death in patients with prostate cancer, using a minimum follow-up of 14 years at one institution, and thus evaluate the minimum life-expectancy for eligibility for radical surgery, radiotherapy and, implicitly, prostate specific antigen (PSA) screening. PATIENTS AND METHODS: The tumour registry of the authors' institution was searched for the records of patients with prostate cancer (stages T1 and nonmetastatic T2-3) over the period 1976-1983, chosen to give a maximum follow-up with sufficient numbers of patients. Kaplan-Meier curves and the mean time to death to 1995 for palpable and impalpable cancers were calculated. Deaths not from cancer and from unknown causes were censored. Patients still alive were also censored, except for in the calculation of mean time to death. RESULTS: Patients with both stages of disease had a steep increase in mortality at 16 years. The mean (SEM) time to prostate cancer-specific death for T1 disease was 17 (1.8) years and for T2-T3 (palpable) was 11.7 (1.2 years). CONCLUSION: These results suggest that, if there is to be one at all, the upper age limit for prostate cancer screening should be 62 years.

Effect of oxygen on radiation-induced DNA damage in isolated nuclei.

In intact mammalian cells, ionizing radiation causes substantially less damage to DNA in the absence of oxygen than in the presence of oxygen. In contrast, when DNA is isolated (usually from viruses) and irradiated in solution, the absence of oxygen does not lead to a decrease in damage unless low-molecular-weight thiols are also present. We investigated an intermediate condition: that of DNA irradiated in isolated nuclei. Using an HPLC-based assay of thiols with electrochemical detection, we have determined that the nuclear isolation procedure leads to the elimination of virtually all low-molecular-weight thiols (predominantly glutathione and cysteine). Thus it was our expectation that the thiol-depleted state would concurrently eliminate the OER, and thereby mimic the isolated DNA system, while retaining structural characteristics of chromosomal DNA. We evaluated radiation-induced DNA damage in isolated nuclei by measuring single-strand breaks using alkaline elution and by measuring double-strand breaks using neutral elution and pulsed-field gel electrophoresis. Despite the removal of low-molecular-weight thiol compounds, the oxygen dependence of radiation-induced damage more closely paralleled that of whole cells than that of DNA in solution. Thus damage of DNA irradiated in isolated nuclei is dependent on oxygen.

Radiation-sensitive tyrosine phosphorylation of cellular proteins: sensitive to changes in GSH content induced by pretreatment with N-acetyl-L-cysteine or L-buthionine-S,R-sulfoximine.

PURPOSE: At relatively high concentrations, ie., > 20 mM, N-acetyl-L-cysteine (NAC) scavenges reactive oxygen species produced by ionizing radiation in aqueous solution. Therefore, the ability of NAC to block signal transduction reactions in vivo, has lead to the suggestion that ROS are necessary for the normal propagation of these signals. In this paper we investigate the mechanism by which NAC alters signal transduction in whole cells. RESULTS: Exposing CHO-K1 cells to ionizing radiation results in elevated pp59fyn kinase activity. Moreover, we observe changes in the phosphotyrosine content of multiple cellular proteins, including one prominent phosphotyrosyl protein with a Mr of 85 kDa. Both the radiation-induced changes in pp59fyn kinase activity and the changes in phosphotyrosine content of pp85 were not affected by exposing K1 cells to NAC during the time of irradiation, suggesting that ROS generated extracellularly are not involved in the radiation-induced changes observed in phosphotyrosyl proteins. We also demonstrate that the cell membrane is an effective barrier against negatively charged NAC. Therefore, it seems unlikely that NAC's ability to block signal transduction reactions is related to scavenging of ROS intracellularly. Chronic exposure, ie., 1 h, to 20 mM NAC lead to a twofold elevation in GSH levels and resulted in a 17% decrease in the phosphotyrosine content of pp85 after exposure to 10 Gy. Moreover, pretreatment with L-buthionine-S,R-sulfoximine (BSO) decreased GSH levels and resulted in elevated phosphotyrosine levels in pp85 isolated from irradiated CHO-K1 cells. CONCLUSIONS: Since many signaling molecules contain redox sensitive cysteine residues that regulate enzyme activity, we suggest that the effects of NAC on radiation-induced signal transduction are due to its ability to alter the intracellular reducing environment, and not related to direct scavenging of ROS.

A novel macrolactam-trisaccharide antifungal antibiotic. Taxonomy, fermentation, isolation, physico-chemical properties, structure elucidation and biological activity.

A novel secondary metabolite SCH 42282 (1), with antifungal activity was isolated from the fermentation broth of a soil actinomycete identified as a Microtetraspora sp. The active compound was separated from the fermentation broth by butanol extraction and purified on a silica gel column and by multi-coil counter current chromatography. The compound was identified as a novel macrolactam trisaccharide related to SCH 38518 (4). The structure was established by hydrolysis of the parent compound and spectroscopic studies of the acetate derivative. The compound is active against Candida spp. (Geometric Mean MIC's. 18 micrograms/ml) but less active SCH 42729 (3). the disaccharide (Geometric Mean MIC's, > or = 10.7 micrograms/ml and SCH 38518 (4), the monosaccharide (Geometric Mean Mic's, 3.8 micrograms/ml.

Analysis of tumor thiol concentrations: comparison of flow cytometric with chemical and biochemical techniques.

The importance of glutathione (GSH) in contributing to cancer therapy resistance is well established. Various advantages may accrue from the ability to determine the distribution of GSH content in individual tumor cells disaggregated from solid tumors using flow cytometric techniques compared with biochemical or chemical measurements of the average GSH level in bulk tissue samples. The flow cytometric technique requires a thiol-reactive fluorescent adduct which is stable and which can differentiate cellular GSH from protein thiols. Thiol-reactive compounds specific for GSH require facilitated conjugation by endogenous cellular enzymes, but such compounds have not been found to accurately monitor GSH in human cells. Compounds which react generally with all thiols require a GSH-depleted calibration control to assess GSH vs. non-GSH components of fluorescent-adduct formation. Our report addresses this question, and compares three different thiol assays in several cell lines. We have found a simple way to control for non-GSH adduct formation. This involves a selective permeabilization process to release low molecular weight adducts (dominated by GSH and cysteine). In application to the desired goal of assessing the distribution of GSH in cells disaggregated from tumors, we have identified a problem with cell-line-specific thiol loss during the tumor cell disaggregation process.

A family of novel macrocyclic lactones, the saccharocarcins produced by Saccharothrix aerocolonigenes subsp. antibiotica. I. Taxonomy, fermentation, isolation and biological properties.

A nocardioform actinomycete, SCC 1886, isolated from a soil sample collected in Ohio was found to produce, in fermentation, six novel macrocyclic lactones, the saccharocarcins. The producing culture was identified as Saccharothrix aerocolonigenes subsp. antibiotica based on the formation of fragmenting substrate mycelia, aerial mycelia that coalesce to form aerial colonies, whole-cell hydrolysates that contain meso-diaminopimelic acid, galactose and rhamnose and physiological comparisons to type species of the genus. Peak production of the saccharocarcins occurred after 95 hours of fermentation in a starch rich medium. The compounds were isolated from the fermentation broth by solvent extraction and purified by HPLC. Isolated compounds were active against Micrococcus luteus, Staphylococcus aureus and Chlamydia trachomatis; none were cytotoxic at concentrations up to 1.0 microgram/ml.