Lan is a high-incidence blood group antigen expressed in more than 99.9% of the population. Identification of the human ABC transporter ABCB6 as the molecular basis of Lan has opened the way for studies assessing the relation of ABCB6 function and expression to health and disease. To date, 34 ABCB6 sequence variants have been described in association with reduced ABCB6 expression based on the genotyping of stored blood showing weak or no reactivity with anti-Lan antibodies. In the present study we examined the red blood cell (RBC) surface expression of ABCB6 by quantitative flow cytometry in a cohort of 47 healthy individuals. Sequencing of the entire coding region of the ABCB6 gene in low RBC ABCB6 expressors identified a new allele (IVS9+1G>A, affecting a putative splice site at the boundary of exon 9) and two nonsynonymous SNPs listed in the SNP database (R192Q (rs150221689) and G588 S (rs145526996)). The R192Q mutation showed co-segregation with reduced RBC ABCB6 expression in a family, and we found the G588 S mutation in a compound heterozygous individual with undetectable ABCB6 expression, suggesting that both mutations result in weak or no expression of ABCB6 on RBCs. Analysis of the intracellular expression pattern in HeLa cells by confocal microscopy indicated that these mutations do not compromise overall expression or the endolysosomal localization of ABCB6. Genotyping of two large cohorts, containing 235 and 1039 unrelated volunteers, confirmed the high allele frequency of Lan-mutations. Our results suggest that genetic variants linked to lower or absent cell surface expression of ABCB6/Langereis may be more common than previously thought.

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Fludarabine, a nucleoside analogue antimetabolite, has complicated pharmacokinetics requiring facilitated transmembrane transport and intracellular conversion to triphosphate nucleotide form (Ara-FATP), causing it to be susceptible to emergence of drug resistance. We are testing a promising strategy to improve its clinical efficacy by direct delivery of Ara-FATP utilizing a biocompatible glycodendrimer nanocarrier system. Here, we present results of a proof-of-concept experiment in several in vitro-cultured leukemic cell lines (CCRF, THP-1, U937) using noncovalent complexes of maltose-modified poly(propyleneimine) dendrimer and fludarabine triphosphate. We show that Ara-FATP has limited cytotoxic activity toward investigated cells relative to free nucleoside (Ara-FA), but complexation with the glycodendrimer (which does not otherwise influence cellular metabolism) drastically increases its toxicity. Moreover, we show that transport via hENT1 is a limiting step in Ara-FA toxicity, while complexation with dendrimer allows Ara-FATP to kill cells even in the presence of a hENT1 inhibitor. Thus, the use of glycodendrimers for drug delivery would allow us to circumvent naturally occurring drug resistance due to decreased transporter activity. Finally, we demonstrate that complex formation does not change the advantageous multifactorial intracellular pharmacodynamics of Ara-FATP, preserving its high capability to inhibit DNA and RNA synthesis and induce apoptosis via the intrinsic pathway. In comparison to other nucleoside analogue drugs, fludarabine is hereby demonstrated to be an optimal candidate for maltose glycodendrimer-mediated drug delivery in antileukemic therapy.

No abstract

Ketamine causes psychotic episodes and is often used as pharmacological model of psychotic-like behavior in animals. There is increasing evidence that molecular mechanism of its action is more complicated than just N-methyl-D-aspartic acid (NMDA) receptor antagonism and involves interaction with the components of calcium homeostatic machinery, in particular plasma membrane calcium pump (PMCA). Therefore, in this study we aimed to characterize brain region-specific effects of ketamine on PMCA activity, interaction with NMDA receptor through postsynaptic density protein 95 (PSD95) scaffolding proteins and glutamate release from nerve endings. In our study, ketamine induced behavioral changes in healthy male rats consistent with psychotic effects. In the same animals, we were able to demonstrate significant inhibition of plasma membrane calcium ATPase (PMCA) activity in cerebellum, hippocampus and striatum. The expression level and isoform composition of PMCAs were also affected in some of these brain compartments, with possible compensatory effects of PMCA1 substituting for decreased expression of PMCA3. Expression of the PDZ domain-containing scaffold protein PSD95 was induced and its association with PMCA4 was higher in most brain compartments upon ketamine treatment. Moreover, increased PSD95/NMDA receptor direct interaction was also reported, strongly suggesting the formation of multiprotein complexes potentially mediating the effect of ketamine on calcium signaling. We further support this molecular mechanism by showing brain region-specific changes in PSD95/PMCA4 spatial colocalization. We also show that ketamine significantly increases synaptic glutamate release in cortex and striatum (without affecting total tissue glutamate content), inducing the expression of vesicular glutamate transporters and decreasing the expression of membrane glutamate reuptake pump excitatory amino acid transporters 2 (EAAT2). Thus, ketamine-mediated PMCA inhibition, by decreasing total Ca2+ clearing potency, may locally raise cytosolic Ca2+ promoting excessive glutamate release. Regional alterations in glutamate secretion can be further driven by PSD95-mediated spatial recruitment of signaling complexes including glutamate receptors and calcium pumps, representing a novel mechanism of psychogenic action of ketamine.