Prokaryote multidrug efflux proteins of the major facilitator superfamily: amplified expression, purification and characterisation.

In bacterial genomes 3-12% of open reading frames are predicted to encode membrane transport proteins. These proteins can be vital for antibiotic efflux, protein/ toxin secretion, cell nutrition, environmental sensing, ATP synthesis, and other functions. Some, such as the multidrug efflux proteins, are potential targets for the development of new antibacterials and also for applications in biotechnology. In general membrane transport proteins are poorly understood, because of the technical difficulties involved in isolating sufficient protein for elucidation of their structure-activity relationships. We describe a general strategy for the amplified expression, purification and characterisation of prokaryotic multidrug efflux proteins of the 'Major facilitator superfamily' of transport proteins, using the Bacillus subtilis multidrug resistance protein, 'Bmr', as example.

Dose-dependent mutation profile in the c-Ha-ras proto-oncogene of skin tumors in mice initiated with benzo[a]pyrene.

Female CD-1 mice were treated topically with a low (25-50 nmol) or high (800 nmol) dose of benzo[a]pyrene (BP) or acetone vehicle, followed by 5 nmol 12-O-tetradecanoylphorbol 13-acetate (TPA) twice a week for 26 weeks. Selective UV radiation fractionation followed by PCR methods were used to analyze histologically defined subsets of cells (approximately 100-200 cells) on formalin-fixed, paraffin-embedded and H&E stained microscope sections. DNA samples from normal-appearing, hyperplastic or tumor regions from the skin of animals from each treatment group were isolated and amplified by PCR with c-Ha-ras-specific primers. Single-strand conformation polymorphism (SSCP) analyses were performed on both exon 1 and 2 products from each sample. DNA extracted from each aberrant band of SSCP analyses was amplified by PCR for further sequence analysis. The data indicate that c-Ha-ras mutations can be detected in normal-looking and hyperplastic epidermal cells as well as in tumor cells obtained from mice initiated with BP and promoted with TPA. The frequencies of c-Ha-ras mutations for normal-looking, hyperplastic and tumor samples were 3/20 (15%), 8/17 (47%) and 58/68 (85%), respectively, for the low dose group and 8/18 (44%), 10/20 (50%) and 64/86 (74%), respectively, for the high dose group. These observations indicate that there were no dose dependencies in the mutation frequencies for normal-looking, hyperplastic and tumor samples. For combined high dose and low dose samples, differences in mutation frequencies of the c-Ha-ras gene between the normal-looking, hyperplastic and tumor samples were highly significant (P < 0.0001, Fisher's exact test). All mutations detected were located at codons 12, 13 and 61 of the c-Ha-ras gene. With the numbers in parentheses indicating the nucleotide position in the coding sequence of the c-Ha-ras proto-oncogene, the distributions of mutations for G-->A (35), G-->T (35), G-->C (37), G-->T (38), C-->A (181), A-->T (182) and A-->G (182) in the low dose tumors were 5, 2, 11, 74, 0, 7 and 2%, respectively, and the distribution of mutations in tumors from animals treated with a high dose of BP were 3, 7, 13, 61, 15, 1 and 0%, respectively. Differences in the global mutation spectra (site and kind of all mutations) for the c-Ha-ras gene between the high and low dose group tumors were statistically significant (P < 0.004, Fisher's exact test) and the major difference between these two groups was C-->A (181) base substitutions. In summary, our data indicate that: (i) 79% of the BP/TPA skin tumors in CD-1 mice had c-Ha-ras mutations for the combined data for high dose and low dose tumors; (ii) the major mutations detected in BP/TPA skin tumors were G-->T transversions; (iii) the global mutation profile in the c-Ha-ras proto-oncogene in skin tumors obtained after initiation with a low dose of BP was different from that obtained after initiation with a high dose of BP.

Combination therapy with stavudine (d4T) plus didanosine (ddI) in children with human immunodeficiency virus infection. The Pediatric AIDS Clinical Trials Group 327 Team.

OBJECTIVES: To evaluate the safety, tolerance, and antiviral activity of combination therapy with stavudine (d4T) plus didanosine (ddI) in symptomatic human immunodeficiency virus (HIV)-infected children. METHODS: The study enrolled HIV-infected children who successfully completed Pediatric AIDS Clinical Trials Group (PACTG) protocol 240 (d4T versus zidovudine [ZDV] monotherapy) without disease progression or who had received ZDV monotherapy by prescription for at least the preceding 6 months. Children who had received d4T monotherapy in PACTG 240 were assigned to treatment with d4T plus ddI (arm 1). Children who had received ZDV monotherapy in PACTG 240 or by prescription were randomized in a double-blind manner to treatment with either d4T alone (arm 2) or d4T plus ddI (arm 3). Patients were followed for 48 weeks each. RESULTS: A total of 108 children were enrolled. The mean age was 5.0 years (range, 1. 6 to 11.5 years), with mean baseline plasma HIV RNA concentration and CD4(+) lymphocyte count of 4.6 log10 copies/mL (range, 2.6 to 5. 9 log10 copies/mL) and 819 cells/microL (range, 8 to 3431 cells/microL), respectively. Both d4T monotherapy and d4T plus ddI combination therapy were well-tolerated, with 96 (89%) patients completing 48 weeks of study treatment. Plasma HIV RNA concentrations showed larger average declines in arm 3 compared with arm 2 at study week 12 (0.49 vs 0.18 log10 copies/mL, respectively); these average declines were maintained through week 48 (0.51 vs 0.17 log10 copies/mL, respectively). Fewer than 8% of the patients in any of the treatment arms had plasma HIV RNA concentrations below the limit of quantification (200 copies/mL) at any time point. CONCLUSIONS: Combination therapy with d4T plus ddI is safe and well-tolerated in HIV-infected children, producing durable, but incomplete, suppression of virus replication. This combination of nucleoside antiretroviral agents may provide a valuable backbone to protease inhibitor-containing treatment regimens for HIV-infected children.

Pharmacokinetics of nevirapine in human immunodeficiency virus type 1-infected pregnant women and their neonates. Pediatric AIDS Clinical Trials Group Protocol 250 Team.

The safety, toxicity, and pharmacokinetics of intrapartum and early newborn nevirapine were evaluated in 17 human immunodeficiency virus type 1-infected women in labor and their newborns. No adverse effects of nevirapine were noted in any study mothers or infants. Following maternal dosing with 200 mg during labor, concentrations exceeding 100 ng/mL (10 times the in vitro IC50) were achieved in the newborns. Nevirapine elimination was prolonged in both mothers and infants, with median half-lives ranging from 36.8 to 65.7 h. Administration of 200 mg orally to the mothers in labor and of a single 2-mg/kg oral dose to the infants at 48-72 h after birth maintained serum concentrations in the infants > 100 ng/mL through 7 days of life.

A randomized comparative trial of stavudine (d4T) versus zidovudine (ZDV, AZT) in children with human immunodeficiency virus infection. AIDS Clinical Trials Group 240 Team.

OBJECTIVES: To compare the safety and tolerance of stavudine (d4T) versus zidovudine (ZDV, AZT) in symptomatic human immunodeficiency virus-infected children 3 months to 6 years of age. METHODS: In an initially double-blind trial, 212 evaluable human immunodeficiency virus-infected children who had received no more than 6 weeks of previous antiretroviral therapy were randomized to receive either d4T (1 mg/kg orally every 12 hours, maximum 40 mg orally every 12 hours) or zidovudine (180 mg/m2 orally every 6 hours, maximum 200 mg orally every 6 hours). The study was unblinded after a median follow-up period of 6.3 months; median follow-up at study closure was 17.3 months. Tolerance, safety, disease progression, and immunologic responses were evaluated. RESULTS: The patient population was young (median age, 1.2 years; range, 0.3 to 6.4 years), with a median baseline CD4+ lymphocyte count of 965 cells/microL (range, 18 to 4238 cells/microL). Neutropenia < 400/microL occurred significantly more commonly among zidovudine recipients (1-year event rates of 20% both up to the time of unblinding and throughout the entire study) than among children receiving d4T (1-year event rates of 5% up to the time of unblinding and 6% throughout the entire study). In exploratory activity analyses using all data collected until study closure, children treated with d4T showed consistently greater positive changes from baseline in weight-for-age-and-gender z scores. As expected in this population of young children, median absolute CD4+ lymphocyte counts decreased in both treatment groups. Smaller changes from baseline were noted among d4T recipients. CONCLUSIONS: In children between the ages of 3 months and 6 years, d4T and zidovudine are largely comparable in terms of safety and tolerance. Neutropenia occurs significantly less commonly among children treated with d4T. There was evidence that weight gain and absolute CD4+ lymphocyte counts were better maintained in children receiving d4T.

Effect of age and sex on the inducibility of hepatic microsomal 4-nitrophenol UDP-glucuronyltransferase activity in rats.

In this report, we examined the effect of age and sex of Long-Evans rats on the inducibility of 4-nitrophenol glucuronyltransferase activity in liver microsomes of animals treated with phenobarbital, dexamethasone, or 3-methylcholanthrene. Glucuronyltransferase activity in control male liver microsomes was approximately 50-75% higher than that in control female liver microsomes. Treatment with sodium phenobarbital (i.p., 75 mg/kg/day) for 3-4 days increased glucuronyltransferase activity by approximately 100% and 60% in liver microsomes from adult female and male rats, respectively, but did not influence this activity in liver microsomes from immature female and male rats. Treatment with dexamethasone (i.p., 75 mg/kg/day) for 3-4 days increased glucuronyltransferase activity by approximately 4-fold in liver microsomes from immature female rats but markedly decreased this enzyme activity in liver microsomes from adult female rats, adult male rats, and immature male rats. Treatment with 3-methylcholanthrene (i.p., 25 mg/kg/day) for 3-4 days increased glucuronyltransferase activity by approximately 10- to 13-fold in liver microsomes from immature female rats, adult female rats, and adult male rats, but only increased this enzyme activity by approximately 1-fold in liver microsomes from immature male rats. In conclusion, the extent of induction of hepatic microsomal glucuronyltransferase activity for 4-nitrophenol is affected dramatically by the age and sex of the animals.

The global environment monitoring system (GEMS): Some recent developments.

GEMS is an established, operational fact, a fact that has been with us for ten years. In 1975, following the instructions of the UN Conference on the Human Environment, UNEP moved into the field of environmental monitoring by establishing at its Nairobi Headquaters a Programme Activity Centre for GEMS. The role given to this Programme Activity Centre is to co-ordinate the disparate international monitoring activities that are conducted thoughout the world, particulary within the UN system, and to advise the Environment Fund of UNEP on how best to support and stimulate the initiation of new activities or the expansion of ongoing ones through the allocation of financial resources to these activities.The data gathered within the GEMS networks are used for assessment of the state of the environment and its trends, for better, more rational management of the environment and its natural resources. Ten years after it began the United Nations GEMS effort can be seen to be both fully operational and global in scope.