Functional analysis of an inosine-guanosine transporter from Leishmania donovani. The role of conserved residues,aspartate 389 and arginine 393

Equilibrative nucleoside transporters encompass two conserved, charged residues that occur within predicted transmembrane domain 8. To assess the role of these "signature" residues in transporter function, the Asp389 and Arg393 residues within the LdNT2 nucleoside transporter from Leishmania donovani were mutated and the resultant phenotypes evaluated after transfection into Delta ldnt2 parasites. Whereas an R393K mutant retained transporter activity similar to that of wild type LdNT2, the R393L, D389E, and D389N mutations resulted in dramatic losses of transport capability. Tagging the wild type and mutant ldnt2 proteins with green fluorescent protein demonstrated that the D389N and D389E mutants targeted properly to the parasite cell surface and flagellum, whereas the expression of R393L at the cell surface was profoundly compromised. To test whether Asp389 and Arg393 interact, a series of mutants was generated, D389R/R393R, D389D/R393D, and D389R/R393D, within the green fluorescent protein-tagged LdNT2 construct. Although all of these ldnt2 mutants were transport-deficient, D389R/R393D localized properly to the plasma membrane, while neither D389R/R393R nor D389D/R393D could be detected. Moreover, a transport-incompetent D389N/R393N double ldnt2 mutant also localized to the parasite membrane, whereas a D389L/R393L ldnt2 mutant did not, suggesting that an interaction between residues 389 and 393 may be involved in LdNT2 membrane targeting. These studies establish genetically that Asp389 is critical for optimal transporter function and that a positively charged or polar residue at Arg393 is essential for proper expression of LdNT2 at the plasma membrane.     

Enhanced expression and activation of CTP:phosphocholine cytidylyltransferase beta2 during neurite outgrowth

During differentiation neurons increase phospholipid biosynthesis to provide new membrane for neurite growth. We studied the regulation of phosphatidylcholine (PC) biosynthesis during differentiation of two neuronal cell lines: PC12 cells and Neuro2a cells. We hypothesized that in PC12 cells nerve growth factor (NGF) would up-regulate the activity and expression of the rate-limiting enzyme in PC biosynthesis, CTP:phosphocholine cytidylyltransferase (CT). During neurite outgrowth, NGF doubled the amount of cellular PC and CT activity. CTbeta2 mRNA increased within 1 day of NGF application, prior to the formation of visible neurites, and continued to increase during neurite growth. When neurites retracted in response to NGF withdrawal, CTbeta2 mRNA, protein, and CT activity decreased. NGF specifically activated CTbeta2 by promoting its translocation from cytosol to membranes. In contrast, NGF did not alter CTalpha expression or translocation. The increase in both CTbeta2 mRNA and CT activity was inhibited by U0126, an inhibitor of mitogen-activated kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2). In Neuro2a cells, retinoic acid significantly increased CT activity (by 54%) and increased CTbeta2 protein, coincident with neurite outgrowth but did not change CTalpha expression. Together, these data suggest that the CTbeta2 isoform of CT is specifically up-regulated and activated during neuronal differentiation to increase PC biosynthesis for growing neurites.     

Equilibrative nucleoside transporter family members from Leishmania donovani are electrogenic proton symporters

Leishmania donovani express two members of the equilibrative nucleoside transporter family; LdNT1 encoded by two closely related and linked genes, LdNT1.1 and LdNT1.2, that transport adenosine and pyrimidine nucleosides and LdNT2 that transports inosine and guanosine exclusively. LdNT1.1, LdNT1.2, and LdNT2 have been expressed in Xenopus laevis oocytes and found to be electrogenic in the presence of nucleoside ligands for which they mediate transport. Further analysis revealed that ligand uptake and transport currents through LdNT1-type transporters are proton-dependent. In addition to the flux of protons that is coupled to the transport reaction, LdNT1 transporters mediate a variable constitutive proton conductance that is blocked by substrates and dipyridamole. Surprisingly, LdNT1.1 and LdNT1.2 exhibit different electrogenic properties, despite their close sequence homology. This electrophysiological study provides the first demonstration that members of the equilibrative nucleoside transporter family can be electrogenic and establishes that these three permeases, unlike their mammalian counterparts, are probably concentrative rather than facilitative transporters.     

Maturation of dendritic cell 2 phenotype by a helminth glycan uses a Toll-like receptor 4-dependent mechanism

The biology of pathogen-associated molecular patterns (PAMPs) stimulating APCs to differentiate into a Th1-promoting phenotype has been well characterized. Conversely, not a single pathogen product that promotes a Th2 phenotype has been rigorously identified. Strong Th2 responses and dendritic cell 2 maturation are driven by helminth extracts, and carbohydrates have been shown to be responsible for much of this activity. In this study, we show that a helminth carbohydrate, lacto-N-fucopentaose III (LNFPIII) functions as an innate Th2 promoter via its action on murine dendritic cells, with the alpha1-3-linked fucose required for this activity. In contrast to Th1-type PAMPs, which activate extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases, the Th2 PAMP LNFPIII preferentially activates extracellular signal-regulated kinase. Furthermore, the ability of LNFPIII to drive DC2 maturation is dependent on signaling via Toll-like receptor 4. These data support a new understanding of how APCs integrate signaling pathways to produce a Th1- or Th2-promoting phenotype.     

Automated measurement of <Emphasis Type="Italic">Drosophila</Emphasis> wings

Background  

Many studies in evolutionary biology and genetics are limited by the rate at which phenotypic information can be acquired. The wings of Drosophila species are a favorable target for automated analysis because of the many interesting questions in evolution and development that can be addressed with them, and because of their simple structure.     

The paroxysm of Plasmodium vivax malaria

The paroxysms of Plasmodium vivax malaria are antiparasite responses that, although distressing to the human host, almost never impart serious acute pathology. Using plasma and blood cells from P. vivax patients, the cellular and noncellular mediators of these events have been studied ex vivo. The host response during a P. vivax paroxysm was found to involve T cells, monocytes and neutrophils, and the activity, among others, of the pyrogenic cytokines tumor necrosis factor alpha and interleukin 2 in addition to granulocyte macrophage-colony stimulating factor. However, interferon gamma activity, associated with serious acute pathogenesis in other studies on malaria, was absent. Induction of the cytokines active during a P. vivax paroxysm depends upon the presence of parasite products, which are released into the plasma before the paroxysm. Chemical identification of these natural parasite products will be important for our understanding of pathogenesis and protection in malaria.     

Endosomal dynamics of Met determine signaling output

Proteasomal activity is required for Met receptor degradation after acute stimulation with hepatocyte growth factor (HGF). Inhibition of proteasomal activity with lactacystin leads to a block in the endocytic trafficking of Met such that the receptor fails to reach late endosomes/lysosomes, where degradation by acid-dependent proteases takes place (). In this article, we have biochemically determined Met internalization rates from the cell surface and shown that lactacystin does not inhibit the initial HGF-dependent internalization step of Met. Instead, it promotes the recycling pathway from early endosomes at the expense of sorting to late endosomes, thereby ensuring rapid return of internalized Met to the cell surface. We have used this perturbation of Met endosomal sorting by lactacystin to examine the consequences for HGF-dependent signaling outputs. In control cells HGF-dependent receptor autophosphorylation reaches a maximal level over 5-10 min but then attenuates over the ensuing 50 min. Furthermore, Met dephosphorylation can be kinetically dissociated from Met degradation. In lactacystin-treated cells, we observe a failure of Met dephosphorylation as well as Met degradation. Elements of the mitogen-activated protein kinase cascade, downstream of receptor activation, show a normal kinetic profile of phosphorylation, indicating that the mitogen-activated protein kinase pathway can attenuate in the face of sustained receptor activation. The HGF-dependent phosphorylation of a receptor substrate that is localized to clathrin-coated regions of sorting endosomes, Hrs, is dramatically reduced by lactacystin treatment. Reduction of cellular Hrs levels by short interfering RNA modestly retards Met degradation and markedly prevents the attenuation of Met phosphorylation. HGF-dependent Hrs phosphorylation and Met dephosphorylation may provide signatures for retention of the receptor in coated regions of the endosome implicated in sorting to lysosomes.     

Targeting Survivin expression induces cell proliferation defect and subsequent cell death involving mitochondrial pathway in myeloid leukemic cells

Survivin, a member of inhibitor of apoptosis family of proteins, plays important roles in both cell proliferation and cell death. We previously observed that Survivin is overexpressed in leukemic cell lines and blasts from patients with acute myelogenous leukemia (AML). To understand the roles of Survivin in AML and search for new approaches to the treatment of AML, we inhibited Survivin expression in HL-60 cells with a Survivin anti-sense oligonucleotide (sur-AS-ODN) (ISIS 23722). This blocked significant numbers of HL-60 cells in G2/M phase, and halted cell proliferation at 24 hrs and progressing over time. There was only a slight increase in the number of apoptotic cells at 24 hrs compared with cells treated with nonsense oligonucleotide (NS-ODN). At 48 hrs, however, there were significant increases in sub-G1 phase and annexin V+ cells, suggesting that cell division defects caused cell death. This was supported by the finding that a reduction in the Survivin protein by sur-AS-ODN in cells under serum-free medium did not induce G2/M block and cell death compared to cells treated with NS-ODN. The formation of polyploid cells was observed 48 hrs after sur-AS-ODN treatment, as was the activation of caspase 3, which suggested that apoptotic cell death had occurred. The mitochondrial release of cytochrome C and Smac and the nuclear translocation of the apoptosis-inducing factor were also detected. Our results suggest that Survivin is essential for cell cycle progression in leukemic cells. Reduced Survivin expression causes a cell-cycle defect that leads to cell death through a mitochondrial pathway. This finding has potential utility for therapy of patients with AML.