Background: The ATP-binding cassette (ABC) superfamily is one of the largest evolutionarily conserved families of proteins. ABC proteins play key roles in cellular detoxification of endobiotics and xenobiotics. Overexpression of certain ABC proteins, among them the multidrug resistance associated protein (MRP), contributes to drug resistance in organisms ranging from human neoplastic cells to parasitic protozoa. In the present study, thePlasmodium berghei mrpgene (pbmrp) was partially characterized and the predicted protein was classified using bioinformatics in order to explore its putative involvement in drug resistance.Methods: Thepbmrpgene from theP. bergheidrug sensitive, N clone, was sequenced using a PCR strategy. Classification and domain organization of pbMRP were determined with bioinformatics. ThePlasmodium spp.MRPs were aligned and analysed to study their conserved motifs and organization. Gene copy number and organization were determined via Southern blot analysis in both N clone and the chloroquine selected line, RC. Chromosomal Southern blots and RNase protection assays were employed to determine the chromosomal location and expression levels ofpbmrpin blood stages.Results: Thepbmrpgene is a single copy, intronless gene with a predicted open reading frame spanning 5820 nucleotides. Bioinformatic analyses show that this protein has distinctive features characteristic of the ABCC sub-family. Multiple sequence alignments reveal a high degree of conservation in the nucleotide binding and transmembrane domains within the MRPs from thePlasmodium spp.analysed. Expression ofpbmrpwas detected in asexual blood stages. Gene organization, copy number and mRNA expression was similar in both lines studied. A chromosomal translocation was observed in the chloroquine selected RC line, from chromosome 13/14 to chromosome 8, when compared to the drug sensitive N clone.Conclusion: In this study, thepbmrpgene was sequenced and classified as a member of the ABCC sub-family. Multiple sequence alignments reveal that this gene is homologous to thePlasmodium y. yoeliiandPlasmodium knowlesi mrp, and thePlasmodium vivaxandPlasmodium falciparum mrp2genes. There were no differences in gene organization, copy number, or mRNA expression between N clone and the RC line, but a chromosomal translocation ofpbmrpfrom chromosome 13/14 to chromosome 8 was detected in RC.

³¹P nuclear magnetic resonance (NMR) spectroscopy was used noninvasively to measure in vivo changes in intracellular pH and intracellular phosphate metabolites in the brains of rats during supercarbia (P_aco₂ ⩾ 400 mm Hg). Five intubated rats were mechanically ventilated with inspired gas mixtures containing 70% CO₂ and 30% O₂. Supercarbia in the rat was observed to cause a greater reduction in cerebral intracellular pH (pH_i) and increase in Pco₂ than observed in other experiments with rats after 15 min of global ischemia. Complete neurologic and metabolic recovery was observed in these animals, despite an average decrease in pH; of 0.63 ± 0.02 pH unit during supercarbia episodes that raised P_aco₂ to 490 ± 80 mm Hg. No change was observed in cerebral intracellular ATP and only a 25% decrease was detected in phosphocreatine. The concentration of free cerebral intracellular ADP, which can be calculated if one assumes that the creatine kinase reaction is in equilibrium, decreased to approximately one-third of its control value. The calculated threefold decrease in the concentration of free ADP and twofold increase in the cytosolic phosphorylation potential suggest that there is increased intracellular oxygenation during supercarbia. Because a more than fourfold increase in intracellular hydrogen ion concentration was tolerated without apparent clinical injury, we conclude that so long as adequate tissue oxygenation and perfusion are maintained, a severe decrease in intracellular pH need not induce or indicate brain injury.

Background: Sporothrix schenckii is a pathogenic dimorphic fungus of worldwide distribution. It grows in the saprophytic form with hyaline, regularly septated hyphae and pyriform conidia at 25°C and as the yeast or parasitic form at 35°C. Previously, we characterized a calcium/calmodulin kinase in this fungus. Inhibitors of this kinase were observed to inhibit the yeast cell cycle in S. schenckii. Results: The presence of RNA interference (RNAi) mechanism in this fungus was confirmed by the identification of a Dicer-1 homologue in S. schenckii DNA. RNAi technology was used to corroborate the role of calcium/calmodulin kinase I in S. schenckii dimorphism. Yeast cells were transformed with the pSilent-Dual2G (pSD2G) plasmid w/wo inserts of the coding region of the calcium/calmodulin kinase I (sscmk1) gene. Transformants were selected at 35°C using resistance to geneticin. Following transfer to liquid medium at 35°C, RNAi transformants developed as abnormal mycelium clumps and not as yeast cells as would be expected. The level of sscmk1 gene expression in RNAi transformants at 35°C was less than that of cells transformed with the empty pSD2G at this same temperature. Yeast two-hybrid analysis of proteins that interact with SSCMK1 identified a homologue of heat shock protein 90 (HSP90) as interacting with this kinase. Growth of the fungus similar to that of the RNAi transformants was observed in medium with geldanamycin (GdA, 10 μM), an inhibitor of HSP90. Conclusions: Using the RNAi technology we silenced the expression of sscmk1 gene in this fungus. RNAi transformants were unable to grow as yeast cells at 35°C showing decreased tolerance to this temperature. The interaction of SSCMK1 with HSP90, observed using the yeast two-hybrid assay suggests that this kinase is involved in thermotolerance through its interaction with HSP90. SSCMK1 interacted with the C terminal domain of HSP90 where effector proteins and co-chaperones interact. These results confirmed SSCMK1 as an important enzyme involved in the dimorphism of S. schenckii, necessary for the development of the yeast phase of this fungus. Also this study constitutes the first report of the transformation of S. schenckii and the use of RNAi to study gene function in this fungus.

Background: Sporothrix schenckii is a pathogenic, dimorphic fungus, the etiological agent of sporotrichosis, a subcutaneous lymphatic mycosis. Dimorphism in S. schenckii responds to second messengers such as cAMP and calcium, suggesting the possible involvement of a calcium/calmodulin kinase in its regulation. In this study we describe a novel calcium/calmodulin-dependent protein kinase gene in S. schenckii, sscmk1, and the effects of inhibitors of calmodulin and calcium/calmodulin kinases on the yeast to mycelium transition and the yeast cell cycle. Results: Using the PCR homology approach a new member of the calcium/calmodulin kinase family, SSCMK1, was identified in this fungus. The cDNA sequence of sscmk1 revealed an open reading frame of 1,221 nucleotides encoding a 407 amino acid protein with a predicted molecular weight of 45.6 kDa. The genomic sequence of sscmk1 revealed the same ORF interrupted by five introns. Bioinformatic analyses of SSCMK1 showed that this protein had the distinctive features that characterize a calcium/calmodulin protein kinase: a serine/threonine protein kinase domain and a calmodulin-binding domain. When compared to homologues from seven species of filamentous fungi, SSCMK1 showed substantial similarities, except for a large and highly variable region that encompasses positions 330 – 380 of the multiple sequence alignment. Inhibition studies using calmodulin inhibitor W-7, and calcium/calmodulin kinase inhibitors, KN-62 and lavendustin C, were found to inhibit budding by cells induced to re-enter the yeast cell cycle and to favor the yeast to mycelium transition. Conclusion: This study constitutes the first evidence of the presence of a calcium/calmodulin kinase-encoding gene in S. schenckii and its possible involvement as an effector of dimorphism in this fungus. These results suggest that a calcium/calmodulin dependent signaling pathway could be involved in the regulation of dimorphism in this fungus. The results suggest that the calcium/calmodulin kinases of yeasts are evolutionarily distinct from those in filamentous fungi.

A recent NMR study reported the elimination of halothane from the brain of rabbits to be ten times slower than expected, based on known anesthetic solubility and cerebral blood flow. The authors conducted a study in five rats using fluorine nuclear magnetic resonance (NMR) spectroscopy to see if major pharmacokinetic discrepancies are associated with the uptake, maintenance, and elimination of halothane from the brain. The rats underwent a 60-min period of halothane anesthesia. They employed a spatially selective NMR spectroscopy technique known as surface coil "depth-pulsing" to assure that the fluorine NMR signals originated in brain tissue, and not in the scalp, muscle, adipose tissue, and bone marrow that surround the brain. After the inspired anesthetic concentration was decreased to zero, the amplitude of the fluorine NMR signal decreased to 40% of its maximum value within 34 +/- 8.0 minutes (n = 5), rather than after 7 h as in the recent study, where the fluorine signal may have contained substantial contributions from metabolites or tissues outside the brain. Fluorine was barely detectable in all of the animals 90 min after stopping the administration of halothane. The authors' results are in agreement with model calculations and several other investigations.