SummaryIn this study, we followed the genomic, lipidomic and metabolomic changes associated with the selection of miltefosine (MIL) resistance in two clinically derived Leishmania donovani strains with different inherent resistance to antimonial drugs (antimony sensitive strain Sb‐S; and antimony resistant Sb‐R). MIL‐R was easily induced in both strains using the promastigote‐stage, but a significant increase in MIL‐R in the intracellular amastigote compared to the corresponding wild‐type did not occur until promastigotes had adapted to 12.2 μM MIL. A variety of common and strain‐specific genetic changes were discovered in MIL‐adapted parasites, including deletions at the LdMT transporter gene, single‐base mutations and changes in somy. The most obvious lipid changes in MIL‐R promastigotes occurred to phosphatidylcholines and lysophosphatidylcholines and results indicate that the Kennedy pathway is involved in MIL resistance. The inherent Sb resistance of the parasite had an impact on the changes that occurred in MIL‐R parasites, with more genetic changes occurring in Sb‐R compared with Sb‐S parasites. Initial interpretation of the changes identified in this study does not support synergies with Sb‐R in the mechanisms of MIL resistance, though this requires an enhanced understanding of the parasite's biochemical pathways and how they are genetically regulated to be verified fully.

The cell nucleus is remarkable in the complexity of critical functions it must perform, and yet this complexity contrasts sharply with the simplified view of the nuclear interior that has persisted for many years. The nucleus is involved in essentially all aspects of cell function and is directly responsible for such intricate tasks as genome packaging, DNA replication, differential gene transcription, RNA processing, selective RNA transport, and cell division. In recent years, great advances have been made in understanding the biochemistry of many nuclear functions; however, we have just begun to discover how these processes are integrated into nuclear structure. Perhaps the nuclear interior has largely escaped our detailed scrutiny because of the enormous density of its content. Electron microscopic observations have provided evidence for internal structures (for review, see Fakan and Puvion 1980; see below), but the functional significance of much of this structure remains poorly understood. The genome itself is not a linear entity in its functional state, hence it is highly likely that an understanding of how coordinated expression of 50-100,000 genes is effectuated within a cell and organism will require a much fuller appreciation of the organization of the genome and its integration with nuclear function.We have investigated the functional organization of the mammalian interphase nucleus, including a perspective that attempts to relate genomic organization with the spatial arrangement of RNA metabolism. The organization of nuclear DNA is of interest in itself, as is the relationship of RNA processing and transport to nuclear structure, hence, these two components are most frequently explored as two distinct entities which are independent and largely unrelated. However, the nucleolus, in which ribosomal RNA transcription and processing occur, clearly establishes the precedent for the integrated compartmentalization of genes and transcript metabolism; this highly specialized subnuclear compartment is formed by the specific clustering of rRNA genes on ten different human chromosomes (for review, see Sheer et al, 1993). One idea we are exploring is that genome structure may be integrated and specifically organized with respect to other regions of increased RNA metabolism, thereby potentially facilitating the selective transcription, processing, or transport of specific RNAs. To address this hypothesis, we are developing an experimental approach that allows the simultaneous detection and high-resolution Cold Spring Harbor Symposta on Quant~tatwe Bwlogy, Volume LVIII 9 1993 localization of specific DNAs and RNAs, as well as proteins, in relation to one another.Key to this has been the development of nonisotopic in situ hybridization techniques (Manning et al. 1975;Bauman et al. 1980;Langer-Safer et al. 1982;Manuelidis and Ward 1984; for review, see Lawrence 1990) coupled with recent advances in the sensitivity and reproducibility of in situ hybridization procedures for DNA and RNA (Lawrence and Singer 1985; Land...

Leishmaniasis is a disease caused by infection with the protozoan parasite Leishmania, which is responsible for three main types of disease: cutaneous leishmaniasis, visceral leishmaniasis and mucocutaneous leishmaniasis based to the site of infection for the particular species. This presents a major challenge to successful drug treatment, as a drug must not only reach antileishmanial concentrations in infected macrophages, the parasites' host cell, but also reach infected cells in locations specific to the type of disease. In this paper we discuss how studies using Leishmania have contributed to our knowledge on how drug delivery systems can be used to improve drug efficacy and delivery.

Development of exact doses (<100) of Strongyloides venezuelensis third-stage larvae in adult Wistar rats was insignificant (mean proportion of 0·076 of the dose at day 8, n = 16) compared with a homogonic strain of S. ratti (0·538, n = 6; 0·726, n = 6) and heterogonic S. ratti (0·681, n = 6). Newly-weaned Wistars allowed development of a mean proportion of S. venezuelensis of 0·298 (n = 4) compared with 0·013 (n = 4) of the same sample of larvae in adult hosts. Experiments with 75Se-labelled larvae established that S. venezuelensis effectively failed to migrate from skin to intestine in adult animals, while mean proportions of 0·141 (n = 5) and 0·138 (n = 4) of the label was found in the intestines of newly-weaned rats 72 h after skin application. Labelled larvae of homogonic S. ratti migrated equally well in both age groups of host (0·350 and 0·358 in 12- and 3-week-olds respectively). Adult S. venezuelensis transferred surgically to the intestines of previously uninfected full-grown Wistars survived over a 21-day period to the same extent as either strain of S. ratti. Resistance of Wistar rats to S. venezuelensis therefore appears to affect the migratory stage preferentially. S. venezuelensis developed better in mature PVG inbred rats (mean = 0·301, n = 20). Studies of S. ratti showed that infections of both strains initiated by exact (<100) doses in Wistar rats had decayed to insignificance between days 26 and 32. The rate of loss of adults of the heterogonic strain was significantly greater than that for the homogonic. The egg content of worms declined as infection progressed and rats were idiosyncratic in their influence on parasite reproduction from the earliest time of sampling (8 d). It was established that ‘autoinfection’ was an unlikely feature of the biology of homogonic S. ratti following the surgical transfer of 450 first-stage larvae to the intestines of 8 adult Wistar rats. No evidence of infection appeared in the guts of these animals 8 days post-transfer. The significance of these results in terms of the biology of Strongyloides spp. naturally occurring in the rat is discussed.

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