Amino Acid Transporters and Exchangers from the SLC1A Family: Structure,Mechanism and Roles in Physiology and Cancer

The Solute Carrier 1A (SLC1A) family includes two major mammalian transport systems—the alanine serine cysteine transporters (ASCT1-2) and the human glutamate transporters otherwise known as the excitatory amino acid transporters (EAAT1-5). The EAATs play a critical role in maintaining low synaptic concentrations of the major excitatory neurotransmitter glutamate, and hence they have been widely researched over a number of years. More recently, the neutral amino acid exchanger, ASCT2 has garnered attention for its important role in cancer biology and potential as a molecular target for cancer therapy. The nature of this role is still being explored, and several classes of ASCT2 inhibitors have been developed. However none have reached sufficient potency or selectivity for clinical use. Despite their distinct functions in biology, the members of the SLC1A family display structural and functional similarity. Since 2004, available structures of the archaeal homologues Glt_Ph and Glt_Tk have elucidated mechanisms of transport and inhibition common to the family. The recent determination of EAAT1 and ASCT2 structures may be of assistance in future efforts to design efficacious ASCT2 inhibitors. This review will focus on ASCT2, the present state of knowledge on its roles in tumour biology, and how structural biology is being used to progress the development of inhibitors.

     

The tumor suppressor CDKN3 controls mitosis

Mitosis is controlled by a network of kinases and phosphatases. We screened a library of small interfering RNAs against a genome-wide set of phosphatases to comprehensively evaluate the role of human phosphatases in mitosis. We found four candidate spindle checkpoint phosphatases, including the tumor suppressor CDKN3. We show that CDKN3 is essential for normal mitosis and G1/S transition. We demonstrate that subcellular localization of CDKN3 changes throughout the cell cycle. We show that CDKN3 dephosphorylates threonine-161 of CDC2 during mitotic exit and we visualize CDC2^pThr-161 at kinetochores and centrosomes in early mitosis. We performed a phosphokinome-wide mass spectrometry screen to find effectors of the CDKN3-CDC2 signaling axis. We found that one of the identified downstream phosphotargets, CKβ phosphorylated at serine 209, localizes to mitotic centrosomes and controls the spindle checkpoint. Finally, we show that CDKN3 protein is down-regulated in brain tumors. Our findings indicate that CDKN3 controls mitosis through the CDC2 signaling axis. These results have implications for targeted anticancer therapeutics.     

Ionic Currents of Human Trabecular Meshwork Cells from Control and Glaucoma Subjects

Increasing evidence suggests that trabecular meshwork (TM) cells participate in the regulation of intraocular pressure by controlling the rate of filtration of the aqueous humor. Ionic conductances that regulate cell volume and shape have been suggested to play an important role in TM cell volume regulation. Here, we compared ionic currents from TM cells derived from a normal subject (CTM) and from an individual affected by glaucoma (GTM). We found that while the ionic current types were similar, the current amplitudes and percentage of cells endowed with specific current at baseline were different in the two cell lines. Thus, we found that the majority of CTM cells were endowed with a swelling-activated Cl^? current at baseline, whereas in the majority of GTM cells this current was not active at baseline and became activated only after perfusion with a hypotonic solution. An inward rectifier K⁺ current was also more prevalent in CTM than in GTM cells. Our work suggests that disregulation of one or more of these ionic currents may be at the basis of TM cell participation in the development of glaucoma.     

Molecular Mechanism by Which a Potent Hepatitis C Virus NS3-NS4A Protease Inhibitor Overcomes Emergence of Resistance

Although optimizing the resistance profile of an inhibitor can be challenging, it is potentially important for improving the long term effectiveness of antiviral therapy. This work describes our rational approach toward the identification of a macrocyclic acylsulfonamide that is a potent inhibitor of the NS3-NS4A proteases of all hepatitis C virus genotypes and of a panel of genotype 1-resistant variants. The enhanced potency of this compound versus variants D168V and R155K facilitated x-ray determination of the inhibitor-variant complexes. In turn, these structural studies revealed a complex molecular basis of resistance and rationalized how such compounds are able to circumvent these mechanisms.     

Latitude drives diversification in Madagascar's endemic dry forest rodent Eliurus myoxinus (subfamily Nesomyinae)

Numerous hypotheses have been proposed for the historical processes governing the rich endemism of Madagascar's biodiversity. The ‘watershed model’ suggests that drier climates in the recent geological past have resulted in the contraction of forests around major watersheds, thereby defining areas of endemism. We test whether this hypothesis explains phylogeographical patterns in a dry forest‐dependent rodent, Eliurus myoxinus, an endemic species widely distributed through western Madagascar. We sequenced the mitochondrial cytochrome b locus and nuclear introns of the β‐fibrinogen and the growth hormone receptor genes for E. myoxinus. Using a parametric bootstrapping approach, we tested whether the mitochondrial gene tree data fit expectations of local differentiation given the watershed model. We additionally estimated population differentiation and historical demographic parameters, and reconstructed the spatial history of E. myoxinus to highlight spatial and temporal patterns of differentiation. The data do not support the watershed model as a clear explanation for the genetic patterns of diversity within extant E. myoxinus populations. We find striking patterns of latitudinal genetic structure within western Madagascar, and indicate possible roles for environmental and ecological gradients along this axis in generating phylogeographical diversity. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 500–517.     

Insecticidal Activity of Bacillus thuringiensis Cry1Bh1 against Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) and Other Lepidopteran Pests

Bacillus thuringiensis is an important source of insect resistance traits in commercial crops. In an effort to prolong B. thuringiensis trait durability, insect resistance management programs often include combinations of insecticidal proteins that are not cross resistant or have demonstrable differences in their site of action as a means to mitigate the development of resistant insect populations. In this report, we describe the activity spectrum of a novel B. thuringiensis Cry protein, Cry1Bh1, against several lepidopteran pests, including laboratory-selected B. thuringiensis-resistant strains of Ostrinia nubilalis and Heliothis virescens and progeny of field-evolved B. thuringiensis-resistant strains of Plutella xylostella and Spodoptera frugiperda. Cry1Bh1 is active against susceptible and B. thuringiensis-resistant colonies of O. nubilalis, P. xylostella, and H. virescens in laboratory diet-based assays, implying a lack of cross-resistance in these insects. However, Cry1Bh1 is not active against susceptible or Cry1F-resistant S. frugiperda. Further, Cry1Bh1 does not compete with Cry1Fa or Cry1Ab for O. nubilalis midgut brush border membrane binding sites. Cry1Bh1-expressing corn, while not completely resistant to insect damage, provided significantly better leaf protection against Cry1Fa-resistant O. nubilalis than did Cry1Fa-expressing hybrid corn. The lack of cross-resistance with Cry1Ab and Cry1Fa along with independent membrane binding sites in O. nubilalis makes Cry1Bh1 a candidate to further optimize for in-plant resistance to this pest.     

Differential Growth of and Nanoscale TiO2 Accumulation in Tetrahymena thermophila by Direct Feeding versus Trophic Transfer from Pseudomonas aeruginosa

Nanoscale titanium dioxide (TiO₂) is increasingly used in consumer goods and is entering waste streams, thereby exposing and potentially affecting environmental microbes. Protozoans could either take up TiO₂ directly from water and sediments or acquire TiO₂ during bactivory (ingestion of bacteria) of TiO₂-encrusted bacteria. Here, the route of exposure of the ciliated protozoan Tetrahymena thermophila to TiO₂ was varied and the growth of, and uptake and accumulation of TiO₂ by, T. thermophila were measured. While TiO₂ did not affect T. thermophila swimming or cellular morphology, direct TiO₂ exposure in rich growth medium resulted in a lower population yield. When TiO₂ exposure was by bactivory of Pseudomonas aeruginosa, the T. thermophila population yield and growth rate were lower than those that occurred during the bactivory of non-TiO₂-encrusted bacteria. Regardless of the feeding mode, T. thermophila cells internalized TiO₂ into their food vacuoles. Biomagnification of TiO₂ was not observed; this was attributed to the observation that TiO₂ appeared to be unable to cross the food vacuole membrane and enter the cytoplasm. Nevertheless, our findings imply that TiO₂ could be transferred into higher trophic levels within food webs and that the food web could be affected by the decreased growth rate and yield of organisms near the base of the web.     

Antimicrobial resistance in Campylobacter: Susceptibility testing methods and resistance trends

Most Campylobacter infections are self-limiting but antimicrobial treatment (e.g., macrolides, fluoroquinolones) is necessary in severe or prolonged cases. Susceptibility testing continues to play a critical role in guiding therapy and epidemiological monitoring of resistance. The methods of choice for Campylobacter recommended by the Clinical and Laboratory Standards Institute (CLSI) are agar dilution and broth microdilution, while a disk diffusion method was recently standardized by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Macrolides, quinolones, and tetracyclines are among the common antimicrobials recommended for testing. Molecular determination of Campylobacter resistance via DNA sequencing or PCR-based methods has been performed. High levels of resistance to tetracycline and ciprofloxacin are frequently reported by many national surveillance programs, but resistance to erythromycin and gentamicin in Campylobacter jejuni remains low. Nonetheless, variations in susceptibility observed over time underscore the need for continued public health monitoring of Campylobacter resistance from humans, animals, and food.