Isolation and Characterization of a Novel Nucleoside from the Urines of Chronic Myelogenous Leukemia Patients

Abstract

From 24 hour collections of urines of chronic myelogenous leukemia (CML) patients, a novel nucleoside was isolated. It was assigned the structure, 5′-deoxyinosine (I) on the basis of UV, NMR and mass spectrometry and by comparison of the spectral data and HPLC and TLC mobilities with those of the authentic sample. Another nucleoside, 5′-deoxy-5′-methylthioadenosine sulfoxide previously isolated from the urines of immunodeficient children was also found in the urine of a CML patient. Possible origin and significance of both of these nucleosides are discussed.     

Restricted lateral mobility of plasma membrane CD4 impairs HIV-1 envelope glycoprotein mediated fusion

We investigated the effect of receptor mobility on HIV-1 envelope glycoprotein (Env)-triggered fusion using B16 mouse melanoma cells that are engineered to express CD4 and CXCR4 or CCR5. These engineered cells are resistant to fusion mediated CD4-dependent HIV-1 envelope glycoprotein. Receptor mobility was measured by fluorescence recovery after photobleaching (FRAP) using either fluorescently-labeled antibodies or transient expression of GFP-tagged receptors in the cells. No significant differences between B16 and NIH3T3 (fusion-permissive) cells were seen in lateral mobility of CCR5 or lipid probes. By contrast CD4 mobility in B16 cells was about seven-fold reduced compared to its mobility in fusion-permissive NIH3T3 cells. However, a CD4 mutant (RA5) that localizes to non-raft membrane microdomains exhibited a three-fold increased mobility in B16 cells as compared with WT-CD4. Interestingly, the B16 cells expressing the RA5 mutant (but not the wild type CD4) and coreceptors supported HIV-1 Env-mediated fusion. Our data demonstrate that the lateral mobility of CD4 is an important determinant of HIV-1 fusion/entry.     

Biochemical and molecular characterization of flavonoid 7-sulfotransferase from Arabidopsis thaliana

Flavonoid compounds play important roles as flower pigments, stress metabolites formed in response to UV, during pollen germination and for polar auxin transport (Trends Plant Sci. 1 (1996) 377). Flavonoid sulfate esters are common in plants, especially the Asteraceae; however, due to the lack of information regarding the factors that regulate their accumulation, their exact role remains to be elucidated. The biosynthesis of flavonol sulfate esters is catalyzed by a number of position specific flavonol sulfotransferases (STs). An Arabidopsis thaliana database search has allowed us to identify and classify 18 putative ST coding sequences. We report here the cloning and characterization of the AtST3a member of this family that is expressed at early stages of seedling development and in the inflorescence stem and siliques of mature plants. The recombinant AtST3a protein exhibits strict specificity for position 7 of flavonoids. In contrast to previously characterized flavonol 7-ST from Flaveria bidentis that sulfonates only flavonol disulfates, AtST3a was found to accept as substrates a number of flavonols and flavone aglycones, as well as their monosulfate esters. The discovery of a flavonol ST from A. thaliana suggests that flavonol sulfates are more widely distributed than originally believed and this model plant could be used to study their biological significance.     

Mouse fat storage‐inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants

Fat storage‐inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)‐localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. Here, we tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension‐cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids in leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. When expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER‐LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ER‐vesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. These results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes.     

Syntheses and antifungal activities of novel 3-amido bearing pseudomycin analogues

As a result of our core SAR effort, we discovered a large number of 3-amido pseudomycin B (PSB) analogues (e.g., 4e LY448212 and 5b LY448731) that retain good in vitro and in vivo (IP) activities against Candida and Cryptococcus without inherent tail vein irritation. Several dimethylamino termini bearing 3-amides (e.g., 5b) also exhibited improved potency against Aspergillus in vitro. When evaluated in a two-week rat toxicology study, it was found that all animals receiving 4e (up to 75 mg/kg) were found to be normal. On the basis of these observations, we are convinced that it is possible to broaden the antifungal spectrum and improve the safety profile of pseudomycin analogues at the same time.     

Crystal structure and association behaviour of the GluR2 amino‐terminal domain

Fast excitatory neurotransmission is mediated largely by ionotropic glutamate receptors (iGluRs), tetrameric, ligand‐gated ion channel proteins comprised of three subfamilies, AMPA, kainate and NMDA receptors, with each subfamily sharing a common, modular‐domain architecture. For all receptor subfamilies, active channels are exclusively formed by assemblages of subunits within the same subfamily, a molecular process principally encoded by the amino‐terminal domain (ATD). However, the molecular basis by which the ATD guides subfamily‐specific receptor assembly is not known. Here we show that AMPA receptor GluR1‐ and GluR2‐ATDs form tightly associated dimers and, by the analysis of crystal structures of the GluR2‐ATD, propose mechanisms by which the ATD guides subfamily‐specific receptor assembly.     

The role of glycosphingolipids in HIV signaling, entry and pathogenesis

Although HIV uses CD4 and coreceptors (CCR5 and CXCR4) for productive infection of T cells, glycosphingolipids (GSL) may play ancillary roles in lymphoid and non-lymphoid cells. Interactions of the HIV Envelope Glycoprotein (Env) with GSL may help HIV in various steps of its pathogenesis. Physical-chemical aspects of the interactions between HIV Env and GSL leading to CD4-dependent entry into lymphocytes, the role of GSL in HIV transcytosis, and CD4-independent entry into non-lymphoid cells are reviewed. An overview of signaling properties of HIV receptors is provided with some speculation on how GSL may play a role in these events by virtue of being in membrane rafts. Finally, we summarize how interactions between HIV and coreceptors leading to signaling and/or fusion can be analyzed by the use of various tyrosine kinase and cytoskeletal inhibitors. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

The HIV Env-mediated fusion reaction

The current general model of HIV viral entry involves the binding of the trimeric viral envelope glycoprotein gp120/gp41 to cell surface receptor CD4 and chemokine co-receptor CXCR4 or CCR5, which triggers conformational changes in the envelope proteins. Gp120 then dissociates from gp41, allowing for the fusion peptide to be inserted into the target membrane and the pre-hairpin configuration of the ectodomain to form. The C-terminal heptad repeat region and the leucine/isoleucine zipper region then form the thermostable six-helix coiled-coil, which drives the membrane merger and eventual fusion. This model needs updating, as there has been a wealth of data produced in the last few years concerning HIV entry, including target cell dependencies, fusion kinetic data, and conformational intermediates. A more complete model must include the involvement of membrane microdomains, actin polymerization, glycosphingolipids, and possibly CD4 and chemokine signaling in entry. In addition, kinetic experiments involving the addition of fusion inhibitors have revealed some of the rate-limiting steps in this process, adding a temporal component to the model. A review of these data that may require an updated version of the original model is presented here.     

Modulation of entry of enveloped viruses by cholesterol and sphingolipids (Review)

Enveloped animal viruses infect host cells by fusion of viral and target membranes. This crucial fusion event occurs either with the plasma membrane of the host cells at the physiological pH or with the endosomal membranes at low pH and is triggered by specific glycoproteins in the virus envelope. Both lipids and proteins play critical and co-operative roles in the fusion process. Interactions of viral proteins with their receptors direct which membranes fuse and viral fusion proteins then drive the process. These fusion proteins operate on lipid assemblies, whose physical and mechanical properties are equally important to the proper functioning of the process. Lipids contribute to the viral fusion process by virtue of their distinct chemical structure, composition and/or their preferred partitioning into specific microdomains in the plasma membrane called 'rafts'. An involvement of lipid rafts in viral entry and membrane fusion has been examined recently. However, the mechanism(s) by which lipids as dynamic raft components control viral envelope-glycoprotein-triggered fusion is not clear. This paper will review literature findings on the contribution of the two raft-associated lipids, cholesterol and sphingolipids in viral entry.