New insights into polyene macrolide biosynthesis in Couchioplanes caeruleus.

Couchioplanes caeruleus DSM43634 synthesises 67-121C, an aromatic heptaene macrolide that contains a mannosyl-mycosaminyl disaccharide. An improved draft genome sequence was used to obtain the biosynthetic gene cluster for this antifungal. Bioinformatic analysis of the polyketide synthase indicated that extension modules 7 and 8 contain A-type ketoreductase and dehydratase domains. These modules are therefore predicted to form cis double bonds. The deduced stereostructure of the 67-121C macrolactone is identical to that experimentally determined for the partricin subgroup of aromatic heptaenes. Some of these polyenes are N-methylated on the aminoacetophenone moiety. The C. caeruleus AceS protein was shown to methylate 4-aminoacetophenone and esters of 4-aminobenzoate, but not 4-aminobenzoate. This suggests that the substrate specificity of AceS prevents it from interfering with folate biosynthesis. The methyltransferase should be valuable for chemoenzymatic alkylation of compounds that contain aminobenzoyl moieties.

Engaging parliamentarians as advocates for women's health: findings from Kenya and Namibia.

Members of parliament (MPs) are well placed to promote national health policies that improve women's access to quality health care, including HIV services. To catalyse political will and leadership, the International Centre for Research on Women, Centre for the Study of AIDS at the University of Pretoria, International Community of Women Living with HIV/AIDS and Realising Rights: The Ethical Globalization Initiative, conducted the Parliamentarians for Women's Health project in select African countries. This paper focus on participatory community assessments - a methodology used by the project to improve MPs' understanding of women's health issues, particularly HIV/AIDS, and to increase their engagement with civil society in order to better represent women's health needs and concerns. In-depth interviews with eight MPs from Kenya and Namibia highlight the value of the assessments in identifying women's health problems and service gaps. The MPs reported that they undertook various activities after the assessments, including gathering more information about women's health from local communities, pushing for new parliamentary committees to be a platform for health issues, using the information from the assessments to inform policy, more carefully reviewing budget allocations and establishing relationships with civil society. Participatory methods can be used to meet political leaders' needs for information and communities' needs to influence policymaking that affects their lives.

Polyene antibiotic biosynthesis gene clusters.

Over the past 15 years the biosynthetic gene clusters for numerous bioactive polyketides have been intensively studied and recently this work has been extended to the antifungal polyene macrolides. These compounds consist of large macrolactone rings that have a characteristic series of conjugated double bonds, as well as an exocyclic carboxyl group and an unusual mycosamine sugar. The biosynthetic gene clusters for nystatin, pimaricin, amphotericin and candicidin have been investigated in detail. These clusters contain the largest modular polyketide synthase genes reported to date. This body of work also provides insights into the enzymes catalysing the unusual post-polyketide modifications, and the genes regulating antibiotic biosynthesis. The sequences also provide clues about the evolutionary origins of polyene biosynthetic genes. Successful genetic manipulation of the producing organisms leading to production of polyene analogues indicates good prospects for generating improved antifungal compounds via genetic engineering.

Effect of liquid feeding weaned pigs on growth performance to harvest.

Four experiments were undertaken to examine the effect of feeding postweaning diets as dry pelleted feed, fresh liquid feed, acidified liquid feed, and fermented liquid feed on pig performance from weaning (26 d) to harvest. In Exp. 1 (n = 12 replicates) and 2 (n = 10 replicates), the treatments were 1) dry pelleted feed and 2) fresh liquid feed. In Exp. 1, 2 kg of starter diet (16.7 MJ of DE/kg and 1.6% lysine) per pig and 5 kg of transition diet (16.7 MJ of DE/kg and 1.5% lysine) per pig followed by a weaner diet (14.0 MJ of DE/kg and 1.36% lysine) were offered to 27 d after weaning. In Exp. 3 (n = 8 replicates), the treatments were 1) dry pelleted feed, 2) fresh liquid feed, and 3) acidified liquid feed. In Exp. 4 (n = 8 replicates), the treatments were 1) dry pelleted feed, 2) acidified liquid feed, and 3) fermented liquid feed. In Exp. 2, 3, and 4, 3 kg of starter diet (16.1 MJ of DE/kg and 1.74% lysine) per pig and 6 kg of transition diet (15.3 MJ of DE/kg and 1.5% lysine) per pig followed by a weaner diet (14.0 MJ of DE/kg and 1.36% lysine) was offered to 27 d after weaning. All treatments were balanced for boars and gilts and diets were offered for ad libitum consumption. Acidified liquid feed was produced by adding lactic acid to the liquid feed so that its pH was decreased to 4.0. Fermented liquid feed was produced by adding an inoculum of Lactococcus lactis subsp. cremoris 303 (1.3%, vol/wt) to the first mix. In Exp. 1, ADG from weaning to d 27 after weaning was 338 and 286 g/d (SEM = 10; P < 0.01) and DM gain/feed in the same period was 888 and 594 g/kg (SEM = 23.1; P < 0.001) for dry pelleted feed and fresh liquid feed, respectively. In Exp. 2, ADG was 391 and 352 g/d (SEM = 6.4; P < 0.01) and DM gain/feed was 856 and 642 g/kg (SEM = 9.9; P < 0.001) for dry pelleted feed and fresh liquid feed, respectively, during the period from weaning to d 27 after weaning. In Exp. 3, ADG was 408, 416, and 433 g/d (SEM = 12.7; P > 0.05) and DM gain/feed was 865, 755, and 789 g/ kg (SEM = 14.5; P < 0.001) for dry pelleted feed, fresh liquid feed, and acidified liquid feed, respectively. In Exp. 4, ADG was 361, 389, and 347 g/d (SEM = 13.2; P = 0.11) and DM gain/feed was 888, 749, and 733 g/ kg (SEM = 15.8; P < 0.001) for dry pelleted feed, acidified liquid feed, and fermented liquid feed, respectively, during the period from weaning to d 27 after weaning. It is concluded that although feeding acidified liquid feed may have some merit in the first 27 d after weaning, this benefit is lost in the subsequent period. No benefit arose from feeding fresh liquid feed or fermented liquid feed. Growth performance from d 28 after weaning to harvest was not improved by any liquid feed treatment.

A prevention strategy for circumventing drug resistance in cancer chemotherapy.

The development of drug resistance is considered to be a major cause for the failure of chemotherapy in a number of types of cancer, including ovarian, breast and lung. Most previous research has focused on approaches to reverse drug resistance once it has arisen, that is, on the use of agents which can make drug-resistant tumors more sensitive to chemotherapy. Unfortunately, this approach has thus far met with only limited clinical success. Because of the prevalence of drug resistance in cases of advanced cancer, there exists an urgent need to develop new approaches to dealing with this problem. We have hypothesized the feasibility of an alternative approach: the use of specific agents to prevent the development of resistance before it arises. Our initial studies to examine this hypothesis have focused on ovarian cancer. We have designed both in vitro and in vivo systems in which resistance develops rapidly after exposure of tumor cells or xenografts to melphalan or cisplatin. Using these systems we have shown that two selenium compounds, selenite and selenomethionine are able to prevent the induction of resistance. Furthermore, inclusion of selenite in a chemotherapeutic protocol can result in a significant enhancement of the efficacy of cisplatin in suppressing the growth of human ovarian tumor xenografts. These results have supported the idea that prevention may be a useful new approach to the problem of drug resistance in cancer chemotherapy.

Amphotericin biosynthesis in Streptomyces nodosus: deductions from analysis of polyketide synthase and late genes.

BACKGROUND: The polyene macrolide amphotericin B is produced by Streptomyces nodosus ATCC14899. Amphotericin B is a potent antifungal antibiotic and has activity against some viruses, protozoans and prions. Treatment of systemic fungal infections with amphotericin B is complicated by its low water-solubility and side effects which include severe nephrotoxicity. Analogues with improved properties could be generated by manipulating amphotericin biosynthetic genes in S. nodosus. RESULTS: A large polyketide synthase gene cluster was cloned from total cellular DNA of S. nodosus. Nucleotide sequence analysis of 113193 bp of this region revealed six large polyketide synthase genes as well as genes for two cytochrome P450 enzymes, two ABC transporter proteins, and genes involved in biosynthesis and attachment of mycosamine. Phage KC515-mediated gene disruption was used to show that this region is involved in amphotericin production. CONCLUSIONS: The availability of these genes and the development of a method for gene disruption and replacement in S. nodosus should allow production of novel amphotericins. A panel of analogues could lead to identification of derivatives with increased solubility, improved biological activity and reduced toxicity.

The eating quality of meat of steers fed grass and/or concentrates.

The objective was to determine, relative to animals expressing their full potential for carcass growth, the impact on meat quality of increasing carcass growth of grazing steers by supplementing with concentrates or by increasing grass supply. Sixty-six continental (Limousin and Charolais) crossbred steers (567 kg) were assigned to one of six diets: (1) 18 kg grass dry matter (DM); (2) 18 kg grass DM grass and 2.5 kg concentrate; (3) 18 kg grass DM and 5 kg concentrate; (4) 6 kg grass DM and 5 kg concentrate; (5) 12 kg grass DM and 2.5 kg concentrate; or (6) concentrates daily. Animals were slaughtered after an average of 95 days. Samples of the M. longissmus dorsi (LD) were collected at the 8-9th rib interface and subjected to sensory analysis and to other assessments of quality following 2, 7, or 14 days aging. Carcass weight gain averaged 360, 631, 727, 617, 551 and 809 g/day for treatments 1 to 6, respectively. There was no difference between diets for colour, Warner-Bratzler shear force (WBSF) or any sensory attribute of the LD. WBSF was negatively correlated with (P<0.05) carcass growth rate (-0.31) but only a small proportion of the variation in meat quality between animals could be attributed to diet pre-slaughter or carcass fatness. It is concluded that high carcass growth can be achieved on a grass-based diet without a deleterious effect on meat quality.

Fatty acid composition, including conjugated linoleic acid, of intramuscular fat from steers offered grazed grass, grass silage, or concentrate-based diets.

The effects of grazed grass, grass silage, or concentrates on fatty acid composition and conjugated linoleic acid (cis-9, trans-11-18:2; CLA) concentrations of i.m. fat of steers fed to achieve similar carcass growth rates were investigated. Fifty steers were divided into 10 blocks based on body weight and assigned at random from within blocks to one of five dietary treatments. The experimental rations offered daily for 85 d preceding slaughter were 1) grass silage for ad libitum intake plus 4 kg of concentrate, 2) 8 kg of concentrate plus 1 kg of hay, 3) 6 kg of grazed grass DM plus 5 kg of concentrate, 4) 12 kg of grazed grass DM plus 2.5 kg concentrate, or 5) 22 kg of grazed grass DM. The concentration of polyunsaturated fatty acids (PUFA) in i.m. fat was higher (P < .05) for steers offered ration 5 than for those given any other ration. Decreasing the proportion of concentrate in the diet, which effectively increased grass intake, caused a linear decrease in the concentration of i.m. saturated fatty acids (SFA) (P < .01) and in the n-6:n-3 PUFA ratio (P < .001) and a linear increase in the PUFA:SFA ratio (P < .01) and the conjugated linoleic acid concentration (P < .001). The data indicate that i.m. fatty acid composition of beef can be improved from a human health perspective by inclusion of grass in the diet.

Lactic acidosis associated with nucleoside reverse transcriptase inhibitors.

Limitations on antiretroviral therapies as a result of resistance and adverse events have been described in the literature. Lactic acidosis has been reported in patients on nucleoside reverse transcriptase inhibitors on rare occasions. Successful assessment and treatment of lactic acidosis is dependent on the nurse's role in the recognition of this condition. This article provides an overview of the clinical presentation, diagnosis, treatment strategies and nursing management of lactic acidosis in patients with HIV/AIDS.

Selenium compounds prevent the induction of drug resistance by cisplatin in human ovarian tumor xenografts in vivo.

PURPOSE: The development of drug resistance is a major cause for the failure of chemotherapy, particularly in ovarian cancer. Most previous research has focused on approaches to reverse drug resistance once it has arisen, that is, on the use of agents which can make drug-resistant tumors more sensitive to chemotherapy. We have suggested the feasibility of an alternative approach: the use of specific agents to prevent the development of resistance. METHODS: We designed an in vivo system to assay for the ability of compounds to prevent the induction of resistance by cisplatin. In this system, mice bearing tumors (which originated from A2780 human ovarian tumor cells) were treated with a low dose (2.6 mg/kg) of cisplatin and the tumors rapidly developed resistance to subsequent cisplatin treatment. Cell lines initiated from these tumors retained the resistant phenotype even after several months in culture. RESULTS: When either selenite or selenomethionine were administered (i.p., 1.5 mg/kg) close to the time of the initial cisplatin treatment, the induction of resistance was prevented. Similar treatments with sulfite or methionine had no effect on the induction of resistance by cisplatin. Studies in cells from treated tumors have indicated that the selenium compounds may prevent the induction of resistance by preventing a cisplatin-induced increase in glutathione level. CONCLUSIONS: Selenium compounds specifically prevent the induction by cisplatin of drug resistance in human ovarian tumors in vivo.

Meat quality of steers finished on autumn grass, grass silage or concentrate-based diets.

The objective of this experiment was to determine the effect of varying the proportions of autumn grass and concentrates and grass silage and concentrates on the quality of meat from cattle with similar rates of carcass growth. Fifty continental crossbred steers were assigned to five treatments. The experimental diets offered were (1) grass silage ad libitum plus 4 kg concentrate (SC), (2) 1 kg hay plus 8 kg concentrate (CO), (3) 6 kg grass dry matter (DM) plus 5 kg concentrate (CG), (4) 12 kg grass DM plus 2.5 kg concentrate (GC) and (5) 22 kg grass DM (GO). The experiment lasted 85 days after which all animals were slaughtered. The right side m. longissmus dorsi was excised from all animals 24 h post slaughter for assessment of meat quality. Treatments SC and CO resulted in animals with whiter (P<0.05) subcutaneous and kidney/channel fat than all other treatments. There was an interaction (P<0.05) between ageing time and treatment with treatment GC having higher (P<0.05) tenderness, texture and acceptability values after 2 days ageing, but not after 7 or 14 days ageing. It is concluded that supplementing grass with low levels of concentrate produced the most tender and acceptable meat at 2 days post mortem, but that further ageing eliminated all treatment effects on eating quality of beef.

Rapid development of glutathione-S-transferase-dependent drug resistance in vitro and its prevention by ethacrynic acid.

Exposure of A2780 human ovarian tumor cells to a low concentration of melphalan in vitro for 7 days resulted in the development of melphalan resistance. This resistance was not a stable characteristic of the cells since it was lost after 2 weeks in culture in the absence of drug. The melphalan-resistant cells exhibited significant cross-resistance to cisplatin but only minor cross-resistance to doxorubicin. The resistant cells had elevated levels of glutathione-S-transferase activity and mRNA. Exposure of the cells to the ethacrynic acid resulted in a decrease in enzyme activity as well as a reversal of their drug-resistant phenotype, indicating that the enzyme is involved in the resistance. When ethacrynic acid was present during the 7-day exposure of the cells to melphalan, the development of drug resistance was prevented. This system may serve as a useful preliminary step in screening for agents which can prevent the development of chemotherapy-induced drug resistance in human cancer.

Treatment of human ovarian tumor xenografts with selenite prevents the melphalan-induced development of drug resistance.

Human ovarian tumors (derived from A2780 cells), growing as subcutaneous xenografts in immunodeficient mice, develop melphalan-resistant cells after a single treatment of the tumor-bearing animal with the drug [Caffrey, Zhang and Frenkel, submitted for publication]. Treatment of the animals with selenite by i.p. injection prevented the development of primary resistance to melphalan as well as cross-resistance to cisplatin. Selenite treatment also prevented the melphalan-induced increase in the cellular level of glutathione. In contrast, selenite administered s.c. or in drinking water had relatively little effect on the development of resistance. The results in this animal model suggest that selenite may be clinically useful in preventing the development of drug resistance during chemotherapy of cancer.

Rapid development of drug resistance in human ovarian tumor xenografts after a single treatment with melphalan in Vivo.

Human ovarian tumors from A2780 cells were grown as xenografts in immunodeficient mice, treated with a single i.p. dose of melphalan and tumor cells were removed and placed into tissue culture. The cells from the treated tumors exhibited an approximately 2-fold resistance to melphalan in vitro compared to cells taken from untreated tumors. This degree of resistance was similar to that of cells from tumors formed from melphalan-resistant A2780-ME cells. The cells from the treated tumors were also resistant to cisplatin but not to doxorubicin. They contained approximately 2-fold higher levels of glutathione than cells from the untreated tumors. Exposure of the cells to buthionine sulfoximine (a specific inhibitor of glutathione biosynthesis) eliminated the difference in glutathione levels as well as the difference in sensitivity to melphalan. When tumor-bearing animals were treated with buthionine sulfoximine in addition to melphalan the resulting tumor cells were not resistant to the drug. Resistance could also be demonstrated in the tumors themselves in vivo: the growth of previously untreated tumors was severely inhibited by a high dose of melphalan (11.7 mg/kg) administered i.p. to the animals, whereas the growth of tumors which had received prior treatment with melphalan was unaffected by the subsequent high dose. The rapid development of drug-resistant tumor cells after a single drug treatment in vivo makes this an excellent system for the investigation of the mechanisms by which resistance develops as well as for use in the screening for agents which can prevent it.

Prevention of the development of melphalan resistance in vitro by selenite.

Exposure of A2780 human ovarian tumor cells to a low concentration of melphalan in vitro for 7 d results in the development of melphalan resistance, which is dependent on elevated cellular levels of glutathione and glutathione S-transferase. The inclusion of selenite (at concentrations as low as 0.2 microM) during the exposure to melphalan completely prevented the development of resistance. Selenite did not prevent the melphalan-induced increase in glutathione, but it did prevent the increase in the activity of glutathione S-transferase. It also prevented the increase in the expression of the glutathione S-transferase gene, suggesting that this may be the mechanism by which it prevents the development of melphalan resistance. The results of this in vitro study suggest that selenite may prove to be useful in preventing the development of drug resistance in vivo.

Sensitivity of melphalan-resistant tumors to selenite in vivo.

Previous studies have demonstrated that melphalan-resistant human ovarian tumor cells exhibit a higher degree of sensitivity to the cytotoxic effects of selenite in vitro than comparable drug-sensitive cells (P.B. Caffrey, G.D. Frenkel, Selenite cytotoxicity in drug resistant and non-resistant human ovarian tumor cells, Cancer Res. 52 (1992) 4812-4816; P.B. Caffrey, G.D. Frenkel, The development of drug resistance by tumor cells in vitro is accompanied by the development of sensitivity to selenite, Cancer Lett. 81 (1994) 59-65). We have now examined the sensitivity of drug-resistant tumors to selenite in vivo. A2780 human ovarian tumor cells, or their melphalan-resistant derivative (A2780ME) cells were injected subcutaneously into nude mice and the resulting tumors were found to be melphalan-sensitive and -resistant, respectively, in vivo. Treatment with selenite (2 mg/kg Se s.c.), which had no overt toxic effect on the animals, resulted in a significant decrease in the rate of growth of the melphalan-resistant tumors, but not on the rate of growth of the drug-sensitive tumors. Thus, melphalan-resistant ovarian tumors are also more sensitive to selenite treatment in vivo.

Evidence for a double-helical structure for modular polyketide synthases.

Modular polyketide synthases are multienzymes responsible for the biosynthesis of a large number of clinically important natural products. They contain multiple sets, or modules, of enzymatic activities, distributed between a few giant multienzymes and there is one module for every successive cycle of polyketide chain extension. We show here that each multienzyme in a typical modular polyketide synthase forms a (possibly helical) parallel dimer, and that each pair of identical modules interacts closely across the dimer interface. Such an arrangement would allow identical modules to share active sites for chain extension, and thus to function independently of flanking modules, which would have important implications both for mechanisms of evolution of polyketide synthases and for their future genetic engineering.

Alcohol and Drug use by Students on a University Campus in Thailand.

This survey was conducted with 501 students from a university in eastern Thailand. The questionnaire was based on the Core Alcohol and Drug Survey designed to survey U.S. higher-education students throughout the United States. It has been translated into Thai language with some modifications in content. Stratified cluster sampling was done based on year in attendance and Faculty (School) affiliation. Two-thirds of the respondents were female. Results showed that males are more involved in alcohol and drug use than females and suffer more consequences as a result. Although Thai students do not use these addictive substances as frequently as U.S. students, there is still cause for concern regarding alcohol use. The number of family members reported by students as having a problem with alcohol or drugs is especially significant.

The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin.

The macrocyclic polyketides rapamycin and FK506 are potent immunosuppressants that prevent T-cell proliferation through specific binding to intracellular protein receptors (immunophilins). The cloning and specific alteration of the biosynthetic genes for these polyketides might allow the biosynthesis of clinically valuable analogues. We report here that three clustered polyketide synthase genes responsible for rapamycin biosynthesis in Streptomyces hygroscopicus together encode 14 homologous sets of enzyme activities (modules), each catalyzing a specific round of chain elongation. An adjacent gene encodes a pipecolate-incorporating enzyme, which completes the macrocycle. The total of 70 constituent active sites makes this the most complex multienzyme system identified so far. The DNA region sequenced (107.3 kbp) contains 24 additional open reading frames, some of which code for proteins governing other key steps in rapamycin biosynthesis.

The development of drug resistance by tumor cells in vitro is accompanied by the development of sensitivity to selenite.

The effects of selenite on cell viability and proliferation in a line of drug-sensitive human ovarian tumor (A2780) cells were compared with its effects on a melphalan-resistant derivative of these cells (A2780-ME) which had been developed in vitro (Hamilton et al. (1985) Biochemical Pharmacol., 34, 2583-2586). With the A2780-ME cells there was a 50% decrease in the number of viable cells (i.e. which exclude Trypan Blue dye) after exposure to less than 100 microM selenite for 6 h. In contrast, exposure to more than 300 microM selenite was required to achieve the same effect in the parent line. Similarly, exposure to 10 microM selenite resulted in a 50% decrease in A2780-ME cell proliferation, whereas this treatment had only a small inhibitory effect on proliferation of the parent cells. Thus, the development of melphalan resistance in vitro was accompanied by the development of selenite sensitivity. Pre-exposure of the two cell types to buthionine sulfoximine eliminated the difference in their intracellular glutathione levels, as well as most of their differential sensitivity to selenite. Furthermore, the two cell types did not exhibit a difference in sensitivity to selenodiglutathione, the product of the reaction of selenite with glutathione. Thus, the increase in intracellular glutathione, which has been shown to be responsible for the development of drug resistance in these cells is also responsible for the development of selenite sensitivity.