WORLD's HIV University is not alone.

AIDS: W.O.R.L.D.'s HIV University (HIVU), sponsored by Glaxo Wellcome, focuses on providing treatment education to women in communities where such information is often lacking. In September 1999, 10 new teams of women from around the country were trained on how to implement an HIVU in their communities. The training had three phases: an overview of steps necessary to implement an HIVU; basic skills-building workshops dealing with issues such as grant writing and group dynamics; and hands-on practice in leading part of an HIVU. The names of the women taking part in the training are provided.^ieng

Transition mutations in the 23S rRNA of erythromycin-resistant isolates of Mycoplasma pneumoniae.

Erythromycin is the drug of choice for treatment of Mycoplasma pneumoniae infections due to its susceptibility to low levels of this antibiotic. After exposure of susceptible strains to erythromycin in vitro and in vivo, mutants resistant to erythromycin and other macrolides were isolated. Their phenotypes have been characterized, but the genetic basis for resistance has never been determined. We isolated two resistant mutants (M129-ER1 and M129-ER2) by growing M. pneumoniae M129 on agar containing different amounts of erythromycin. In broth dilution tests both strains displayed resistance to high levels of several macrolide-lincosamide-streptogramin B (MLS) antibiotics. In binding studies, ribosomes isolated from the resistant strains exhibited significantly lower affinity for [14C]erythromycin than did ribosomes from the M129 parent strain. Sequencing of DNA amplified from the region of the 2S rRNA gene encoding domain V revealed an A-to-G transition in the central loop at position 2063 of M129-ER1 and a similar A-to-G transition at position 2064 in M129-ER2. Transitions at homologous locations in the 23S rRNA from other organisms have been shown to result in resistance to MLS antibiotics. Thus, MLS-like resistance can occur in M. pneumoniae as the result of point mutations in the 23S rRNA gene which reduce the affinity of these antibiotics for the ribosome. Since they involve only single-base changes, development of resistance to erythromycin in vivo by these mechanisms could be relatively frequent event.

Genetic map of the Mycoplasma genitalium chromosome.

At 600 kb, the genome of Mycoplasma genitalium is among the smallest known for cellular organisms capable of independent replication. As such, elucidation of the genetic makeup and chromosome architecture of this organism is of considerable interest. We have located 631 markers on the physical map of M. genitalium. The clones have been mapped by hybridizing 20 overlapping cosmid and lambda clones which encompass the entire M. genitalium chromosome to replica filters containing 856 genomic DNA clones. Three hundred fifty-six of these clones represent sequence tag sites, which were previously characterized by database searches. The remaining markers represent clones with an average size of 2.5 kb derived from Sau3A1 partial digestion of genomic DNA. The hybridization data can be divided into three classes: clones which hybridized to only one cosmid; clones which hybridized to two adjacent and overlapping cosmids; and clones which hybridized to several cosmids, which represent repetitive DNA. This rapid approach for placing clones on the physical map has allowed useful comparisons to be made with other bacterial chromosomes, especially that of the closely related organism M. pneumoniae, and has provided insight to the types of events which may have led to the reduction in size of this genome. Future use of these data is discussed.

Construction of an ordered genomic library of Mycoplasma genitalium.

As a first step towards sequencing the chromosome of the suspected human pathogen Mycoplasma genitalium, we attempted to clone its entire genome in a set of ordered cosmids. Cosmid libraries were established by partial digestion of M. genitalium genomic DNA with Sau3AI or EcoRI. A chromosome-walking strategy was used to identify 20 overlapping cosmid clones which contained over 99% of the genome. The final 5.1 kb could not be cloned in cosmids, and was eventually obtained from a genomic library established in a lambda vector. Correspondence of cloned and genomic EcoRI fragments indicated no detectable major deletions or rearrangements in the library. The library was oriented on established XhoI and SmaI physical maps of the chromosome with restriction sites present at the expected locations in the library. The genome contained 74 EcoRI fragments which added up to a total genome size of 578 kb. These were arranged in a partial EcoRI physical map, and those containing the MgPa major attachment protein-encoding operon and its repeat sequences were identified. The existence of this ordered genomic library, which accurately and completely encompasses the entire M. genitalium genome, should serve as a valuable tool for many future studies of this organism and facilitate our long-term goal of sequencing its genome.

Novel 6,5-fused ring heterocyclic antifolates: biochemical and biological characterization.

Six novel antifolates with 2,4-diaminopyrimidine-fused five-membered rings containing either pyrrole or cyclopentene rings were characterized at the cellular and biochemical level. Five of these antifolates were more growth inhibitory to the CCRF-CEM human leukemia cell line than methotrexate [MTX; drug concentration effective at inhibiting cell growth by 50% relative to untreated control (EC50), 12 nM], the antifolate used in the clinic, and two were more potent than 10-ethyl-10-deazaaminopterin (EC50, 2.7 nM); similar patterns of response were obtained in the FaDu and A253 squamous carcinoma cell lines. In addition, the growth inhibitory potency of these antifolates was generally less dependent on exposure time than was MTX. Growth inhibitory effects could be reversed by leucovorin, indicating an antifolate mechanism. These antifolates targeted dihydrofolate reductase (DHFR) based on direct human DHFR inhibition assays [drug concentration inhibiting enzyme activity by 50% (IC50), 0.6-28 nM; MTX IC50, 0.8 nM] and the cross-resistance of MTX-resistant CCRF-CEM cells containing elevated DHFR. Inhibition of human thymidylate synthase was generally weak. These 6,5-fused ring heterocyclic antifolates utilized the reduced folate/MTX transporter for uptake, based on the cross-resistance of MTX uptake-impaired CCRF-CEM cells, and were efficient substrates for this uptake system, based on inhibition of [3H]MTX uptake (IC50, 0.3-5.8 microM; aminopterin IC50, 2.6 microM). These analogues were substrates for CCRF-CEM folylpolyglutamate synthetase, with several being among the most active substrates now known (highest Vrel/Km 0.73; MTX and 10-ethyl-10-deazaaminopterin, 0.013 and 0.24, respectively). Substrate activity for murine intestinal folylpolyglutamate synthetase was also assayed, and a different specificity pattern was observed. These new antifolates are apparently not substrates for aldehyde oxidase. Analogues containing the fused cyclopentene ring are preferred to those containing the fused pyrrole ring based on growth inhibitory potency, effectiveness against decreased uptake mutants and apparent affinity for transport, and inhibition of DHFR. In addition, fused cyclopentene-containing analogues are efficiently polyglutamylated. The data indicate that antifolates with 2,4-diaminopyrimidine-fused five-membered rings, especially those containing the fused cyclopentene ring, are an important new class of antifolates which warrant further exploration at the synthetic and preclinical levels.

Cross-resistance studies of folylpolyglutamate synthetase-deficient, methotrexate-resistant CCRF-CEM human leukemia sublines.

CCRF-CEM human leukemia sublines resistant to short-term methotrexate (MTX) exposure as a result of decreased folylpolyglutamate synthetase (FPGS) activity were examined for their response to other cytotoxic agents. The R3/7 and R30dm sublines display 25 and 1%, respectively, of the FPGS activity of CCRF-CEM cells as measured with MTX in vitro. Response to agents in outgrowth experiments was examined under both continuous exposure (120 h, where MTX resistance is not observed) and short-term (6-14.5 h) exposure. During continuous exposure to various classes of agents, cross-resistance of R3/7 and R30dm that correlated with FPGS level was not observed, although some minor (< or = 3-fold) stochastic variations in sensitivity were noted. These agents included actinomycin D, Adriamycin, etoposide, vincristine, cisplatin, cytosine arabinoside, 5-fluorouracil, and some other antifolates. Cross-resistance during continuous exposure that did correlate with FPGS level was noted, however, to glutamate-containing thymidylate synthase inhibitors (including ICI D1694) and, to a minor extent, to 6-mercaptopurine and 5-fluorodeoxyuridine. Slight collateral sensitivity during continuous exposure that apparently correlated with FPGS level was noted to the lipid-soluble antifolate trimetrexate and to 5,8-dideazapteroyl-L-ornithine, an FPGS-specific inhibitor. In short-term exposures (where MTX resistance of the sublines is observed), the resistant sublines displayed sensitivity or cross-resistance to each agent that was qualitatively similar to that observed for the same agent in continuous exposure. Because of the requirement for reduced folates in the anti-DNA mechanism of action of fluoropyrimidines and the current clinical use of leucovorin (LV) to enhance their effects, the interaction of LV and fluoropyrimidines was examined. The results suggest that even highly FPGS-deficient cells are as sensitive to the effects of LV modulation as are wild-type cells even at fluoropyrimidine exposure times as short as 4 h.