Effect of glucantime on field and patient isolates of New World Leishmania: use of growth parameters of promastigotes to assess antimony susceptibility.

The effect of pentavalent meglumine antimoniate (glucantime) on the growth kinetics of promastigotes of 13 South American Leishmania strains isolated from patients, sylvatic reservoirs, and vectors was studied. Four of five L. (Viannia) braziliensis human isolates were obtained from drug-responsive patients and one was isolated from an unresponsive mucocutaneous-type infection. Studies involved the cell yield at the late log phase, the growth rate, and the growth-curve patterns of promastigotes in vitro. Restriction-fragment-length polymorphism, pulsed-field gel electrophoresis, and hybridization with the gene coding for a P-glycoprotein from L. (V.) guyanensis were used in an attempt to correlate the resistance phenotype with gene amplification. Consistent differences observed in both cell yield and growth rate among the isolates in the presence of glucantime indicated these parameters to be good criteria for the estimation of susceptibility to glucantime. Drug susceptibility varied widely between strains, indicating a great genetic heterogeneity of the isolates. Five L. (V.) braziliensis strains and three L. (V.) guyanensis strains were shown to be susceptible to glucantime. Five isolates were resistant, four of which were obtained from sylvatic vectors and one, from a patient with an unresponsive mucocutaneous infection. Molecular analyses of total DNA indicated the presence of a pgpA-related gene in all strains tested. No amplified sequence could be detected, suggesting that pgpA amplification is not involved in glucantime resistance in these wild Leishmania strains.

Cell surface carbohydrates and in vivo infectivity of glucantime-sensitive and resistant Leishmania (Viannia) guyjanensis cell lines.

The cell surface plays an important role in the interaction of parasites with their hosts. Drug resistance in the protozoan Leishmania may involve changes in cell-surface composition, although it is not known whether infectivity is also affected. One sensitive and two glucantime-resistant lines of Leishmania (Viannia) guyanensis previously isolated were inoculated into hamsters. The sensitive line caused the disease to manifest earlier than the resistant lines. Imprinting analyses of infected macrophages showed that the sensitive line was more infective than the resistant cell lines. In vitro drug resistance was evaluated and the comparative analyses of dose-response curves showed that the susceptibility pattern of the sensitive line did not change after passage in animals, but a decrease in drug resistance was observed in resistant cell lines recovered from the mammalian host. Cell surface carbohydrates of sensitive and resistant cell lines were analysed before and after passage in animals by agglutination tests with several plant lectins. Passage in animals changed the agglutination pattern for many lectins from all three cell lines. Loss of reactivity to lectins seemed to be correlated with a decrease in infectivity of the parasite-resistant cell lines. This study opens possibilities for exploring the relationship between drug susceptibility, infectivity and surface carbohydrate composition of protozoan parasites.

Leishmania (V.) guyanensis: isolation and characterization of glucantime-resistant cell lines.

A glucantime sensitive Leishmania (V.) guyanensis strain was used to obtain in vitro resistant cell lines, by increments in glucantime concentrations employing both one step and stepwise protocols. Whereas the effective concentration of drug that inhibited the growth of wild type cells by 50% (EC50 value) was 0.20 mg Sb(v)/mL, the resistant cells were able to grow in glucantime concentrations greater than 8.0 mg/mL. The resistant cell lines were partially characterized by their in vitro response to glucantime, the stability of resistance phenotype, cross resistance to a range of drugs, and also by the analysis of total DNA fragments generated by restriction endonucleases and blot hybridization. Amplified DNA sequence similar to a P-glycoprotein analog from Leishmania tarentolae (ltpgpA gene) was observed in all the resistant cell lines obtained through the one-step protocol. These cell lines showed cross resistance to heavy metals but were sensitive to puromycin, vinblastine, and pentostam.

Relationship of the methylenetetrahydrofolate reductase C677T polymorphism with microsatellite instability and promoter hypermethylation in sporadic colorectal cancer

The methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism is associated with the expression of a thermolabile enzyme with decreased activity that influences the pool of methyl-donor molecules. Several studies have reported an association between C677T polymorphism and susceptibility to colorectal cancer (CRC). Considering that methylation abnormalities appear to be important for the pathogenesis of CRC, we examined the correlation between the genotype of the MTHFR C677T polymorphism, hypermethylation of the promoter region of five relevant genes (DAPK, MGMT, hMLH1, p16(INK4a), and p14(ARF)), and microsatellite instability, in 106 patients with primary CRCs in Brazil. We did not find significant differences in the genotypic frequencies of the MTHFR C677T polymorphism when one or more loci were hypermethylated. However, we did find a significant excess of 677TT individuals among patients with CRC who had microsatellite instability. This strong association was independent of the methylation status of hMLH1 and of the biogeographical genomic ancestry of the patients. Although the mechanism responsible for the link between the C677T polymorphism and microsatellite instability was not apparent, this finding may provide a clue towards a better understanding of the pathogenesis of microsatellite instability in human colorectal cancer.

Mining microorganism EST databases in the quest for new proteins

Microorganisms with large genomes are commonly the subjects of single-round partial sequencing of cDNA, generating expressed sequence tags (ESTs). Usually there is a great distance between gene discovery by EST projects and submission of amino acid sequences to public databases. We analyzed the relationship between available ESTs and protein sequences and used the sequences available in the secondary database, clusters of orthologous groups (COG), to investigate ESTs from eight microorganisms of medical and/or economic relevance, selecting for candidate ESTs that may be further pursued for protein characterization. The organisms chosen were Paracoccidioides brasiliensis, Dictyostelium discoideum, Fusarium graminearum, Plasmodium yoelii, Magnaporthe grisea, Emericella nidulans, Chlamydomonas reinhardtii and Eimeria tenella, which have more than 10,000 ESTs available in dbEST. A total of 77,114 protein sequences from COG were used, corresponding to 3,201 distinct genes. At least 212 of these were capable of identifying candidate ESTs for further studies (E. tenella). This number was extended to over 700 candidate ESTs (C. reinhardtii, F. graminearum). Remarkably, even the organism that presents the highest number of ESTs corresponding to known proteins, P. yoelii, showed a considerable number of candidate ESTs for protein characterization (477). For some organisms, such as P. brasiliensis, M. grisea and F. graminearum, bioinformatics has allowed for automatic annotation of up to about 20 of the ESTs that did not correspond to proteins already characterized in the organism. In conclusion, 4093 ESTs from these eight organisms that are homologous to COG genes were selected as candidates for protein characterization