Biomedical subjects

Peter Rohloff

Publications and source records attributed to Peter Rohloff.

3 recordsLinked to original sources

PubMed2004

Trypanosoma brucei plasma membrane-type Ca(2+)-ATPase 1 (TbPMC1) and 2 (TbPMC2) genes encode functional Ca(2+)-ATPases localized to the acidocalcisomes and plasma membrane, and essential for Ca(2+) homeostasis and growth.

Trypanosoma brucei adaptation and survival in its host involve integrated regulation of Ca(2+) pumps (Ca(2+)-ATPases), which are essential in calcium ion homeostasis. Here we report the cloning and sequencing of two genes (TbPMC1 and TbPMC2) encoding plasma membrane-type Ca(2+)-ATPases (PMCAs) of T. brucei, an agent of African trypanosomiasis. Indirect immunofluorescence analysis using antibodies against the proteins and against epitope tags introduced into each protein showed that TbPMC1 co-localized with the vacuolar H(+)-pyrophosphatase to the acidocalcisomes while TbPMC2 localized to the plasma membrane. Northern and Western blot analyses revealed that TbPMC1 and TbPMC2 are up-regulated during blood stages. TbPMC1 and TbPMC2 suppressed the Ca(2+) hypersensitivity of a mutant of S. cerevisiae that has a defect in vacuolar Ca(2+) accumulation. T. brucei Ca(2+)-ATPase genes were functionally characterized by using double-stranded RNA interference (RNAi) methodology to produce inducible Ca(2+)-ATPase-deficient procyclic forms. Similar results were obtained with bloodstream form trypomastigotes, except that the RNAi system was leaky and mRNA and protein levels recovered with time. The induction of dsRNA (RNAi) caused gross morphological alterations, and growth inhibition of procyclic forms. Induction of RNAi against TbPMC1 but not against TbPMC2 caused elevated levels of cytosolic Ca(2+) and decreased mobilization of Ca(2+) from intracellular stores following ionophore addition. These results establish that T. brucei PMCA-Ca(2+)-ATPases are essential for parasite viability and validate them as targets for drug development.

Amino Acid Sequence↗

PubMed2003

Regulatory volume decrease in Trypanosoma cruzi involves amino acid efflux and changes in intracellular calcium.

A regulatory volume decrease (RVD) in response to hyposmotic stress has been characterized in different life-cycle stages of Trypanosoma cruzi. Hyposmotic stress initially caused swelling, but this was rapidly reversed by a compensatory volume reversal that was essentially complete by 5 min. Volume recovery was associated with an amino acid efflux that accounted for approximately 50% of the regulatory volume decrease in all three life-cycle stages. The amino acid efflux was selective for neutral and anionic amino acids, but excluded cationic amino acids. Acidocalcisomes contained an amino acid pool over four times more concentrated than whole-cell levels, but about 90% of this was composed of Arg and Lys, so involvement of this pool in amino acid efflux was ruled out. Hyposmotic stress induced a rise in intracellular calcium that was dependent on influx of calcium across the plasma membrane, since chelation of extracellular calcium abolished the response. Influx of calcium was confirmed by demonstration of manganese-mediated quenching of intracellular fura-2 fluorescence and partial inhibition of the rise in calcium by calcium channel blockers. Manipulation of intra- and extracellular calcium levels had minor effects on the initial rate of amino acid efflux and no effect on the rate of volume recovery.

Amino Acids↗

PubMed2002

Characterization of isolated acidocalcisomes from Toxoplasma gondii tachyzoites reveals a novel pool of hydrolyzable polyphosphate.

Toxoplasma gondii tachyzoites were fractionated by modification of an iodixanol density gradient method previously used for acidocalcisome isolation from Trypanosoma cruzi epimastigotes. Fractions were characterized using electron microscopy, x-ray microanalysis, and enzymatic markers, and it was demonstrated that the heaviest (pellet) fraction contains electron-dense vacuoles rich in phosphorus, calcium, and magnesium, as found before for acidocalcisomes. Staining with 4',6-diamidino-2-phenylindole (DAPI) indicated that poly- phosphate (polyP) was preferentially localized in this fraction together with pyrophosphate (PP(i)). Using an enzyme-based method, millimolar levels (in terms of P(i) residues) of polyP chains of less than 50 residues long and micromolar levels in polyP chains of about 700-800 residues long were found to be preferentially localized in this fraction. The fraction also contained the pyrophosphatase and polyphosphatase activities characteristic of acidocalcisomes. Western blot analysis using antibodies against proteins from micronemes, dense granules, rhoptries, and plasma membrane showed that the acidocalcisomal fraction was not contaminated by these other organelles. T. gondii polyP levels rapidly decreased upon exposure of the parasites to a calcium ionophore (ionomycin), to an inhibitor of the V-H(+)-ATPase (bafilomycin A(1)), or to the alkalinizing agent NH(4)Cl. These changes were in parallel to an increase in intracellular Ca(2+) concentration, suggesting a close association between polyP hydrolysis and Ca(2+) release from the acidocalcisome. These results provide a useful method for the isolation and characterization of acidocalcisomes, showing that they are distinct from other previously recognized organelles present in T. gondii, and provide evidence for the role of polyP metabolism in response to cellular stress.

Acid Anhydride Hydrolases↗