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We raised a specific antiserum against the recombinant M2 subunit protein of ribonucleotide reductase of Leishmania mexicana amazonensis in rabbit. This antiserum was used to study the expression and cellular location of the M2 protein in wildtype as well as hydroxyurea-resistant variants (HuR) of the parasite. The protein increased with increasing dose of the drug used for selection of resistance. The increase in protein level was accompanied by an increase in the copy numbers of mRNA of the M2 gene in the variants. In contrast to mammalian cells, the M2 protein of Leishmania is located in the nucleus rather than in the cytoplasm. The number of cells expressing M2 protein is also different in mammalian cells versus Leishmania. In mammalian cells, expression of M2 protein is a strictly S-phase-correlated event and in exponentially growing cells only approximately 50% of the cells are in S-phase and only these cells synthesize M2 protein. In L. m. amazonensis, however, almost all exponentially growing cells are positive for M2 protein. This makes it unlikely that M2 protein expression in Leishmania is S-phase dependent. In view of these findings, a fresh look in the future into the regulatory mechanisms of synthesis and the site of action of RNR in L. m. amazonensis is warranted.

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Cellular ATP and rate of respiration are important for the cell survival. We have analyzed both the parameters in wild type and arsenite resistant Leishmania mexicana amazonensis. There was no significant change observed in the rate of respiration and cellular ATP content between drug resistant cells (resistance to 30 microM of sodium arsenite) and wild type cells. Further, we have tested the effect of higher concentrations (i.e. 100 microM and 500 microM) of sodium arsenite on the ATP content of the cells. An elevated level of ATP was observed only in wild type cells after short term exposure (2 h) to 100 microM of the drug, whereas, drug resistant cells initially resist with higher toxic dosage of drug (i.e. 500 microM) but failed to maintain the normal ATP level. In conclusion, respiration and ATP is not a prime event associated with drug resistance in Leishmania. Resistance to metals like arsenic and antimony in Leishmania is multifactorial events.

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An alkaliphilic, endospore-forming bacterium isolated from Brazilian soil was taxonomically studied and is proposed as a new Paenibacillus species. This organism (strain 324T) was particularly distinguishable from other Paenibacillus species by its ability to grow optimally at pH 10 and 40 degrees C. The DNA G+C content was 5.0 mol%. The diamine acid of the cell-wall peptidoglycan was meso-diaminopimelic acid. MK-7 was the predominant menaquinone and anteiso-C15:0 was the major fatty acid. Levels of 16S rDNA similarity between strain 324T and other Paenibacillus species were 90.6-95.9%. Phylogenetically, strain 324T formed an evolutionary lineage distinct from other species within the evolutionary radiation encompassing the genus Paenibacillus. Based on phenotyic and chemotaxonomic properties, and phylogenetic inference, it is proposed that strain 324T should be placed in the genus Paenibacillus as a new species is strain 324T should be placed in the genus Paenibacilus as a new species, Paenibacillus campinasensis. This type strain of the new species is strain 325T (= KCTC 0364BP).

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To explain the low cross-hybridization between kinetoplast DNA maxicircles of Leishmania parasites that show DNA amplification and those of parasites without DNA amplification, we isolated and cloned two maxicircle fragments, one specific to each group of parasites. The cloned fragment from wildtype L. m. amazonensis (MbpW94) and that from an arsenite-resistant variant with DNA amplification (MpbA29) hybridized only to maxicircles from parasites of the group from which the fragment was originally derived. Both fragments were A+T-rich, tandemly repeated, and lacked long conserved open reading frames and transcriptional products. MpbW94 (685 bp) was harbored in a segment of roughly 12 kb in maxicircles of wildtype parasites and of an arsenite-resistant variant without DNA amplification, while MbpA29 (1121 bp) occupied a 6- to 7-kb segment of maxicircle DNA in arsenite- and tunicamycin-resistant variants with DNA amplification. These maxicircle DNA segments appear to resemble previously described maxicircle divergent regions of other kinetoplastids. The presence of these specific sequences allows differentiation between maxicircles of drug-resistant L. m. amazonensis with DNA amplification and those of parasites without DNA amplification and helps explain the low cross-hybridization between maxicircles of the two parasite groups. Furthermore, these sequences allow the study of the kinetics of the changeover of A+T-rich regions of maxicircles during the transition period from one maxicircle type to the other.

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Certain minor minicircle sequence classes in the kinetoplast DNA (kDNA) networks of arsenite- or tunicamycin-resistant Leishmania mexicana amazonensis variants whose nuclear DNA is amplified appear to be preferentially selected to replicate (S. T. Lee, C. Tarn, and K. P. Chang, Mol. Biochem. Parasitol. 58:187-204, 1993). These sequences replace the predominant wild-type minicircle sequences to become dominant species in the kDNA network. The switch from wild-type-specific to variant-specific minicircles takes place rapidly within the same network, the period of minicircle dominance changes being defined as the transition period. To investigate the structural organization of the kDNA networks during this transition period, we analyzed kDNA from whole arsenite-resistant Leishmania parasites by dot hybridization with sequence-specific DNA probes and by electron-microscopic examination of isolated kDNA networks in vitro. Both analyses concluded that during the switch of dominance the predominant wild-type minicircle class was rapidly lost and that selective replication of variant-specific minicircles subsequently filled the network step by step. There was a time during the transition when few wild-type- or variant-specific minicircles were present, leaving the network almost empty and exposing a species of thick, long, fibrous DNA which seemed to form a skeleton for the network. Both minicircles and maxicircles were found to attach to these long DNA fibrils. The nature of the long DNA fibrils is not clear, but they may be important in providing a framework for the network structure and a support for the replication of minicircles and maxicircles.

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