BackgroundThe 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, the Plasmodium berghei mrp gene (pbmrp) was partially characterized and the predicted protein was classified using bioinformatics in order to explore its putative involvement in drug resistance.MethodsThe pbmrp gene from the P. berghei drug sensitive, N clone, was sequenced using a PCR strategy. Classification and domain organization of pbMRP were determined with bioinformatics. The Plasmodium 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 of pbmrp in blood stages.ResultsThe pbmrp gene 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 the Plasmodium spp. analysed. Expression of pbmrp was 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.ConclusionIn this study, the pbmrp gene was sequenced and classified as a member of the ABCC sub-family. Multiple sequence alignments reveal that this gene is homologous to the Plasmodium y. yoelii and Plasmodium knowlesi mrp, and the Plasmodium vivax and Plasmodium falciparum mrp2 genes. There were no differences in gene organization, copy number, or mRNA expression between N clone and the RC line, but a chromosomal translocation of pbmrp from chromosome 13/14 to chromosome 8 was detected in RC.

BackgroundSporothrix 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.ResultsThe 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.ConclusionsUsing 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.

BackgroundSporothrix schenckii is a pathogenic dimorphic fungus, the etiological agent of sporotrichosis, a lymphocutaneous disease that can remain localized or can disseminate, involving joints, lungs, and the central nervous system. Pathogenic fungi use signal transduction pathways to rapidly adapt to changing environmental conditions and S. schenckii is no exception. S. schenckii yeast cells, either proliferate (yeast cell cycle) or engage in a developmental program that includes proliferation accompanied by morphogenesis (yeast to mycelium transition) depending on the environmental conditions. The principal intracellular receptors of environmental signals are the heterotrimeric G proteins, suggesting their involvement in fungal dimorphism and pathogenicity. Identifying these G proteins in fungi and their involvement in protein-protein interactions will help determine their role in signal transduction pathways.ResultsIn this work we describe a new G protein α subunit gene in S. schenckii, ssg-2. The cDNA sequence of ssg-2 revealed a predicted open reading frame of 1,065 nucleotides encoding a 355 amino acids protein with a molecular weight of 40.9 kDa. When used as bait in a yeast two-hybrid assay, a cytoplasmic phospholipase A2 catalytic subunit was identified as interacting with SSG-2. The sspla2 gene, revealed an open reading frame of 2538 bp and encoded an 846 amino acid protein with a calculated molecular weight of 92.62 kDa. The principal features that characterize cPLA2 were identified in this enzyme such as a phospholipase catalytic domain and the characteristic invariable arginine and serine residues. A role for SSPLA2 in the control of dimorphism in S. schenckii is suggested by observing the effects of inhibitors of the enzyme on the yeast cell cycle and the yeast to mycelium transition in this fungus. Phospholipase A2 inhibitors such as AACOCF3 (an analogue of archidonic acid) and isotetrandrine (an inhibitor of G protein PLA2 interactions) were found to inhibit budding by yeasts induced to re-enter the yeast cell cycle and to stimulate the yeast to mycelium transition showing that this enzyme is necessary for the yeast cell cycle.ConclusionA new G protein α subunit gene was characterized in S. schenckii and protein-protein interactions studies revealed this G protein alpha subunit interacts with a cPLA2 homologue. The PLA2 homologue reported here is the first phospholipase identified in S. schenckii and the first time a PLA2 homologue is identified as interacting with a G protein α subunit in a pathogenic dimorphic fungus, establishing a relationship between these G proteins and the pathogenic potential of fungi. This cPLA2 homologue is known to play a role in signal transduction and fungal pathogenesis. Using cPLA2 inhibitors, this enzyme was found to affect dimorphism in S. schenckii and was found to be necessary for the development of the yeast or pathogenic form of the fungus.

Summary: 31p nuclear magnetic resonance (NMR) spec troscopy 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% 02' Supercarbia in the rat was observed to cause a greater reduction in cerebral intracellular pH (pH) and increase in Peo2 than observed in other experiments with rats after 15 min of global ischemia. Complete neurologic and metabolic recovery was observed in these animals, de spite an average decrease in pHj of 0.63 ± 0.02 pH unit during supercarbia episodes that raised Paco2 to 490 ± 80 mm Hg. No change was observed in cerebral intra cellular ATP and only a 25% decrease was detected in phosphocreatine. The concentration of free cerebral in- SUPERCARBIA AND BRAIN ENERGY METABOLISMIschemia and hypoventilation in clinical situa tions can cause hypercarbia in brain cells. During these conditions, some cells usually will also suffer from inadequate oxygen availability. Deleterious consequences of hypercarbia are known to arise from secondary physiological phenomena such as seizures, sympathetic stimulation, and certain ef fects of acidosis, such as cardiac irritability and de creased oxygen availability due to a right shift of the hemoglobin dissociation curve (Nunn, 1977). Abbreviations used: Cr, creatine; NMR, nuclear magnetic res onance; PCr, phosphocreatine; pHa, arterial pH; pHi' cerebral intracellular pH; PME, phosphomonoesters. tracellular ADP, which can be calculated if one assumes that the creatine kinase reaction is in equilibrium, de creased to approximately one-third of its control value. The calculated threefold decrease in the concentration of free ADP and twofold increase in the cytosolic phos phorylation potential suggest that there is increased in tracellular oxygenation during supercarbia. Because a more than fourfold increase in intracellular hydrogen ion concentration was tolerated without apparent clinical in jury, we conclude that so long as adequate tissue oxy genation and perfusion are maintained, a severe decrease in intracellular pH need not induce or indicate brain in jury.

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.