A novel selection regime for differentiation defects demonstrates an essential role for the stumpy form in the life cycle of the African trypanosome. (1/23)

A novel selection scheme has been developed to isolate bloodstream forms of Trypanosoma brucei, which are defective in their ability to differentiate to the procyclic stage. Detailed characterization of one selected cell line (defective in differentiation clone 1 [DiD-1]) has demonstrated that these cells are indistinguishable from the wild-type population in terms of their morphology, cell cycle progression, and biochemical characteristics but are defective in their ability to initiate differentiation to the procyclic form. Although a small proportion of DiD-1 cells remain able to transform, deletion of the genes for glycophosphatidyl inositol-phospholipase C demonstrated that this enzyme was not responsible for this inefficient differentiation. However, the attenuated growth of the Delta-glycophosphatidyl inositol-phospholipase C DiD-1 cells in mice permitted the expression of stumpy characteristics in this previously monomorphic cell line, and concomitantly their ability to differentiate efficiently was restored. Our results indicate that monomorphic cells retain expression of a characteristic of the stumpy form essential for differentiation, and that this is reduced in the defective cells. This approach provides a new route to dissection of the cytological and molecular basis of life cycle progression in the African trypanosome.  (+info)

Unexpected anthracycline-mediated alterations in iron-regulatory protein-RNA-binding activity: the iron and copper complexes of anthracyclines decrease RNA-binding activity. (2/23)

Anthracyclines are effective antineoplastic agents. However, the interaction of these drugs with iron (Fe) is an important cause of myocardial toxicity, limiting their therapeutic use (J Lab Clin Med 122:245-251, 1993). To overcome this limitation, it is crucial to understand how anthracyclines interact with the Fe metabolism of myocardial and neoplastic cells. Iron-regulatory proteins (IRPs) play vital roles in regulating cellular Fe metabolism via their mRNA-binding activity. We showed that doxorubicin (DOX) and its analogs interfere with tumor and myocardial cell Fe metabolism by affecting the RNA-binding activity of IRPs. Unexpectedly, experiments with the free radical scavengers, catalase, superoxide dismutase, ebselen, and Mn(III) tetrakis (4-benzoic acid) porphyrin complex, suggested that the effects of DOX on IRP-RNA-binding activity were not due to anthracycline-mediated free radical production. In contrast to previous studies, we showed that the DOX metabolite, doxorubicinol, had no effect on IRP-RNA-binding activity. Rather, the anthracycline-Fe and -copper (Cu) complexes decreased IRP-RNA-binding activity, indicating that formation of anthracycline-metal complexes may affect cellular Fe metabolism. In addition, anthracyclines prevented the response of IRPs to the depletion of intracellular Fe by chelators. This information may be useful in designing novel therapeutic strategies against tumor cells by combining chelators and anthracyclines. Interestingly, the effect of DOX on primary cultures of cardiomyocytes was similar to that observed using neoplastic cells, and particularly notable was the decrease in IRP2-RNA-binding activity. Our results add significant new information regarding the effects of anthracyclines on Fe metabolism that may lead to the design of more effective treatments.  (+info)

Cold shock and regulation of surface protein trafficking convey sensitization to inducers of stage differentiation in Trypanosoma brucei. (3/23)

Transmission of a protozoan parasite from a vertebrate to invertebrate host is accompanied by cellular differentiation. The signals from the environment that trigger the process are poorly understood. The model parasite Trypanosoma brucei proliferates in the mammalian bloodstream and in the tsetse fly. On ingestion by the tsetse, the trypanosome undergoes a rapid differentiation that is marked by replacement of the variant surface glycoprotein (VSG) coat with GPI-anchored EP and GPEET procyclins. Here we show that a cold shock of DeltaT > 15 degrees C is sufficient to reversibly induce high-level expression of the insect stage-specific EP gene in the mammalian bloodstream stages of T. brucei. The 3'-UTR of the EP mRNA is necessary and sufficient for the increased expression. During cold shock, EP protein accumulates in the endosomal compartment in the proliferating, slender, bloodstream stage, whereas the EP is present on the plasma membrane in the quiescent, stumpy, bloodstream stage. Thus, there is a novel developmentally regulated cell surface access control mechanism for a GPI-anchored protein. In addition to inducing EP expression, cold shock results in the acquisition of sensitivity to micromolar concentrations of cis-aconitate and citrate by stumpy but not slender bloodstream forms. The cis-aconitate and citrate commit stumpy bloodstream cells to differentiation to the procyclic stage along with rapid initial proliferation. We propose a hierarchical model of three events that regulate differentiation after transmission to the tsetse: sensing the temperature change, surface access of a putative receptor, and sensing of a chemical cue.  (+info)

The FAD-dependent tricarballylate dehydrogenase (TcuA) enzyme of Salmonella enterica converts tricarballylate into cis-aconitate. (4/23)

Tricarballylate is the causative agent of grass tetany, a ruminant disease characterized by acute magnesium deficiency. Tricarballylate toxicity has been attributed to its ability to chelate magnesium and to inhibit aconitase, a Krebs cycle enzyme. Neither the ruminant nor the normal rumen flora can catabolize tricarballylate to ameliorate its toxic effects. However, the gram-negative enterobacterium Salmonella enterica can use tricarballylate as a carbon and energy source, providing an opportunity to study the genes and enzymes required for tricarballylate catabolism. The tricarballylate utilization (tcu) genes are organized into two transcriptional units, i.e., tcuR and tcuABC. Here, we report the initial biochemical analysis of TcuA. TcuA catalyzed the oxidation of tricarballylate to cis-aconitate. The apparent K(m) of TcuA for tricarballylate was 3.8 +/- 0.4 mM, with a V(max) of 7.9 +/- 0.3 mM min(-1), turnover number (k(cat)) of 6.7 x 10(-2) s(-1), and a catalytic efficiency (k(cat)/K(m)) of 17.8 M(-1) s(-1). Optimal activity was measured at pH 7.5 and 30 degrees C. The enzyme was inactivated at 45 degrees C. One mole of FAD was present per mole of TcuA. We propose a role for TcuB as an electron shuttle protein responsible for oxidizing FADH(2) back to FAD in TcuA.  (+info)

The three-dimensional crystal structure of the PrpF protein of Shewanella oneidensis complexed with trans-aconitate: insights into its biological function. (5/23)

In bacteria, the dehydration of 2-methylcitrate to yield 2-methylaconitate in the 2-methylcitric acid cycle is catalyzed by a cofactor-less (PrpD) enzyme or by an aconitase-like (AcnD) enzyme. Bacteria that use AcnD also require the function of the PrpF protein, whose function was previously unknown. To gain insights into the function of PrpF, the three-dimensional crystal structure of the PrpF protein from the bacterium Shewanella oneidensis was solved at 2.0 A resolution. The protein fold of PrpF is strikingly similar to those of the non-PLP-dependent diaminopimelate epimerase from Haemophilus influenzae, a putative proline racemase from Brucella melitensis, and to a recently deposited structure of a hypothetical protein from Pseudomonas aeruginosa. Results from in vitro studies show that PrpF isomerizes trans-aconitate to cis-aconitate. It is proposed that PrpF catalysis of the cis-trans isomerization proceeds through a base-catalyzed proton abstraction coupled with a rotation about C2-C3 bond of 2-methylaconitate, and that residue Lys73 is critical for PrpF function. The newly identified function of PrpF as a non-PLP-dependent isomerase, together with the fact that PrpD-containing bacteria do not require PrpF, suggest that the isomer of 2-methylaconitate that serves as a substrate of aconitase must have the same stereochemistry as that synthesized by PrpD. From this, it follows that the 2-methylaconitate isomer generated by AcnD is not a substrate of aconitase, and that PrpF is required to generate the correct isomer. As a consequence, the isomerase activity of PrpF may now be viewed as an integral part of the 2-methylcitric acid cycle.  (+info)

A high-throughput method to measure NaCl and acid taste thresholds in mice. (6/23)

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A surface transporter family conveys the trypanosome differentiation signal. (7/23)

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Synthesis and properties of polycarboxylate-type green surfactants with S- or N-linkages. (8/23)

Polycarboxylate-type green surfactants with either sulfide- (S-) or imino- (N-) linkages were prepared in high yields by a single addition reaction of fatty mercaptan or fatty amine with unsaturated polycarboxylic acids such as fumaric, maleic, itaconic and aconitic acids. They exhibited surfactant properties and excellent biodegradabilities. Also, green surfactants with S-linkages showed better calcium ion sequestration abilities compared to the corresponding surfactant having an N-linkage. Among these surfactants, aconitic acid-derived polycarboxylate with an S-linkage exhibited calcium ion sequestration capacities similar to that of disodium 3-oxapentanedioate (ODA), a conventional calcium ion sequestrant on a molar basis of the surfactant.  (+info)

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