In vitro and in vivo studies have demonstrated that UCB (unconjugated bilirubin) is neurotoxic. Although previous studies suggested that both MRP1 (multidrug resistance-associated protein 1) and MDR1 (multidrug resistance protein 1) may protect cells against accumulation of UCB, direct comparison of their role in UCB transport was never performed. To this end, we used an inducible siRNA (small interfering RNA) expression system to silence the expression of MRP1 and MDR1 in human neuroblastoma SH-SY5Y cells. The effects of in vitro exposure to clinically-relevant levels of unbound UCB were compared between unsilenced (control) cells and cells with similar reductions in the expression of MRP1 or MDR1, documented by RT-PCR (reverse transcription-PCR) (mRNA), immunoblotting (protein), and for MDR1, the enhanced net uptake of a specific fluorescent substrate. Cytotoxicity was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] test. MRP1-deficient cells accumulated significantly more UCB and suffered greater cytotoxicity than controls. By contrast, MDR1-deficient cells exhibited UCB uptake and cytotoxicity comparable with controls. At intermediate levels of silencing, the increased susceptibility to UCB toxicity closely correlated with the decrease in the expression of MRP1, but not of MDR1. These data support the concept that limitation of cellular UCB accumulation, due to UCB export mediated by MRP1, but not MDR1, plays an important role in preventing bilirubin encephalopathy in the newborn.

As part of a study on the malate dehydrogenase isozymes (MDHs) from Trypanosomatids, three different fractions with MDH activity were obtained from crude extracts of Leishmania mexicana promastigotes combining two different chromatographic steps. Gel filtration chromatography in native conditions showed that most of the MDH activity present in the crude extracts eluted in a single peak, which corresponded to a lower apparent molecular mass (≅57kDa) than the value expected for typical MDHs. To further characterize the leishmanial isozymes, three putative MDH genes, presumably corresponding to the mitochondrial, glycosomal and cytosolic isoforms were amplified by PCR, cloned into bacterial expression vectors, and the recombinant enzymes purified. Digitonin extraction of intact L. mexicana promastigotes and immunofluorescence microscopy of L. major promastigotes confirmed the subcellular compartmentation of each of the three isozymes. Western blot analysis showed that the three MDHs are developmentally regulated. At the protein level, these isozymes are remarkably more abundant in amastigotes than in promastigotes of L. mexicana. Altogether our results demonstrate the presence of three MDH isoforms with slightly distinct biochemical properties and different subcellular localization in Leishmania spp. Presumably the functional and biochemical features of these isozymes reflect the metabolic adaptation to the different nutrient sources these parasites have to face along their life cycle. These results also emphasize the differences among Trypanosomatids in this area of metabolism, since in the case of Trypanosoma brucei the cMDH is the only isoform expressed in bloodstream trypomastigotes, whereas in Trypanosoma cruzi cMDH is absent.

Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.