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Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a correctly folded, functional state and can be purified in amounts suitable for structural investigations.

Gemcitabine is a hydrophilic clinical anticancer drug that requires nucleoside transporters to cross plasma membranes and enter cells. Pancreatic adenocarcinomas with low levels of nucleoside transporters are generally resistant to gemcitabine and are currently a clinical problem. We tested whether transfection of human concentrative nucleoside transporter 3 (hCNT3) using ultrasound and lipid stabilized microbubbles could increase gemcitabine uptake and sensitivity in HEK293 cells made nucleoside transport deficient by pharmacologic treatment with dilazep. To our knowledge, no published data exists regarding the utility of using hCNT3 as a therapeutic gene to reverse gemcitabine resistance. Our ultrasound transfection system--capable of transfection of cell cultures, mouse muscle and xenograft CEM/araC tumors--increased hCNT3 mRNA and (3)H-gemcitabine uptake by >2,000- and 3,400-fold, respectively, in dilazep-treated HEK293 cells. Interestingly, HEK293 cells with both functional human equilibrative nucleoside transporters and hCNT3 displayed 5% of (3)H-gemcitabine uptake observed in cells with only functional hCNT3, suggesting that equilibrative nucleoside transporters caused significant efflux of (3)H-gemcitabine. Efflux assays confirmed that dilazep could inhibit the majority of (3)H-gemcitabine efflux from HEK293 cells, suggesting that hENTs were responsible for the majority of efflux from the tested cells. Oocyte uptake transport assays were also performed and provided support for our hypothesis. Gemcitabine uptake and efflux assays were also performed on pancreatic cancer AsPC-1 and MIA PaCa-2 cells with similar results to that of HEK293 cells. Using the MTS proliferation assay, dilazep-treated HEK293 cells demonstrated 13-fold greater resistance to gemcitabine compared to dilazep-untreated HEK293 cells and this resistance could be reversed by transfection of hCNT3 cDNA. We propose that transfection of hCNT3 cDNA using ultrasound and microbubbles may be a method to reverse gemcitabine resistance in pancreatic tumors that have little nucleoside transport activity which are resistant to almost all current anticancer therapies.

Exploration of purinergic signaling in brainstem homeostatic control processes is challenging the traditional view that the biphasic hypoxic ventilatory response, which comprises a rapid initial increase in breathing followed by a slower secondary depression, reflects the interaction between peripheral chemoreceptor-mediated excitation and central inhibition. While controversial, accumulating evidence supports that in addition to peripheral excitation, interactions between central excitatory and inhibitory purinergic mechanisms shape this key homeostatic reflex. The objective of this review is to present our working model of how purinergic signaling modulates the glutamatergic inspiratory synapse in the preBötzinger Complex (key site of inspiratory rhythm generation) to shape the hypoxic ventilatory response. It is based on the perspective that has emerged from decades of analysis of glutamatergic synapses in the hippocampus, where the actions of extracellular ATP are determined by a complex signaling system, the purinome. The purinome involves not only the actions of ATP and adenosine at P2 and P1 receptors, respectively, but diverse families of enzymes and transporters that collectively determine the rate of ATP degradation, adenosine accumulation and adenosine clearance. We summarize current knowledge of the roles played by these different purinergic elements in the hypoxic ventilatory response, often drawing on examples from other brain regions, and look ahead to many unanswered questions and remaining challenges.

Numerous national associations and multiple reviews have documented the safety and efficacy of strength training for children and adolescents. The literature highlights the significant training-induced increases in strength associated with youth strength training. However, the effectiveness of youth strength training programs to improve power measures is not as clear. This discrepancy may be related to training and testing specificity. Most prior youth strength training programs emphasized lower intensity resistance with relatively slow movements. Since power activities typically involve higher intensity, explosive-like contractions with higher angular velocities (e.g., plyometrics), there is a conflict between the training medium and testing measures. This meta-analysis compared strength (e.g., training with resistance or body mass) and power training programs (e.g., plyometric training) on proxies of muscle strength, power, and speed. A systematic literature search using a Boolean Search Strategy was conducted in the electronic databases PubMed, SPORT Discus, Web of Science, and Google Scholar and revealed 652 hits. After perusal of title, abstract, and full text, 107 studies were eligible for inclusion in this systematic review and meta-analysis. The meta-analysis showed small to moderate magnitude changes for training specificity with jump measures. In other words, power training was more effective than strength training for improving youth jump height. For sprint measures, strength training was more effective than power training with youth. Furthermore, strength training exhibited consistently large magnitude changes to lower body strength measures, which contrasted with the generally trivial, small and moderate magnitude training improvements of power training upon lower body strength, sprint and jump measures, respectively. Maturity related inadequacies in eccentric strength and balance might influence the lack of training specificity with the unilateral landings and propulsions associated with sprinting. Based on this meta-analysis, strength training should be incorporated prior to power training in order to establish an adequate foundation of strength for power training activities.

hENT1 (human equilibrative nucleoside transporter 1) is inhibited by nanomolar concentrations of various structurally distinct coronary vasodilator drugs, including dipyridamole, dilazep, draflazine, soluflazine and NBMPR (nitrobenzylmercaptopurine ribonucleoside). When a library of randomly mutated hENT1 cDNAs was screened using a yeast-based functional complementation assay for resistance to dilazep, a clone containing the W29G mutation was identified. Multiple sequence alignments revealed that this residue was highly conserved. Mutations at Trp29 were generated and tested for adenosine transport activity and inhibitor sensitivity. Trp29 mutations significantly reduced the apparent Vmax and/or increased the apparent Km values for adenosine transport. Trp29 mutations increased the IC50 values for hENT1 inhibition by dipyridamole, dilazep, NBMPR, soluflazine and draflazine. NBMPR and soluflazine displayed remarkably similar trends, with large aromatic substitutions at residue 29 resulting in the lowest IC50 values, suggesting that both drugs could interact via ring-stacking interactions with Trp29. The W29T mutant displayed a selective loss of pyrimidine nucleoside transport activity, which contrasts with the previously identified L442I mutant that displayed a selective loss of purine nucleoside transport. W29T, L442I and the double mutant W29T/L442I were characterized kinetically for nucleoside transport activity. A helical wheel projection of TM (transmembrane segment) 1 suggests that Trp29 is positioned close to Met33, implicated previously in nucleoside and inhibitor recognition, and that both residues line the permeant translocation pathway. The data also suggest that Trp29 forms part of, or lies close to, the binding sites for dipyridamole, dilazep, NBMPR, soluflazine and draflazine. [ABSTRACT FROM AUTHOR]

Human and other mammalian concentrative (Na+-linked) nucleoside transport proteins belong to a membrane protein family (CNT, TC 2.A.41) that also includes Escherichia coli H+-dependent nucleoside transport protein NupC. Here, we report the cDNA cloning and functional characterization of a CNT family member from the pathogenic yeast Candida albicans. This 608 amino acid residue H+/nucleoside symporter, designated CaCNT, contains 13 predicted transmembrane domains (TMs), but lacks the exofacial, glycosylated carboxyl-terminus of its mammalian counterparts. When produced in Xenopus oocytes, CaCNT exhibited transport activity for adenosine, uridine, inosine and guanosine but not cytidine, thymidine or the nucleobase hypoxanthine. Apparent Km values were in the range 16–64 µM, with Vmax : Km ratios of 0.58–1.31. CaCNT also accepted purine and uridine analogue nucleoside drugs as permeants, including cordycepin (3'-deoxyadenosine), a nucleoside analogue with anti-fungal activity. Electrophysiological measurements under voltage clamp conditions gave a H+ to [14C]uridine coupling ratio of 1 : 1. CaCNT, obtained from logarithmically growing cells, is the first described cation-coupled nucleoside transporter in yeast, and the first member of the CNT family of proteins to be characterized from a unicellular eukaryotic organism. Copyright © 2003 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]