The molecular basis of folate salvage in Plasmodium falciparum: characterization of two folate transporters

Tetrahydrofolates are essential cofactors for DNA synthesis and methionine metabolism. Malaria parasites are capable both of synthesizing tetrahydrofolates and precursors de novo and of salvaging them from the environment. The biosynthetic route has been studied in some detail over decades, whereas the molecular mechanisms that underpin the salvage pathway lag behind. Here we identify two functional folate transporters (named PfFT1 and PfFT2) and delineate unexpected substrate preferences of the folate salvage pathway in Plasmodium falciparum. Both proteins are localized in the plasma membrane and internal membranes of the parasite intra-erythrocytic stages. Transport substrates include folic acid, folinic acid, the folate precursor p-amino benzoic acid (pABA), and the human folate catabolite pABAG(n). Intriguingly, the major circulating plasma folate, 5-methyltetrahydrofolate, was a poor substrate for transport via PfFT2 and was not transported by PfFT1. Transport of all folates studied was inhibited by probenecid and methotrexate. Growth rescue in Escherichia coli and antifolate antagonism experiments in P. falciparum indicate that functional salvage of 5-methyltetrahydrofolate is detectable but trivial. In fact pABA was the only effective salvage substrate at normal physiological levels. Because pABA is neither synthesized nor required by the human host, pABA metabolism may offer opportunities for chemotherapeutic intervention.     

Unfolded protein response is required in nu/nu mice microvasculature for treating breast tumor with tunicamycin

Up-regulation of the dolichol pathway, a "hallmark" of asparagine-linked protein glycosylation, enhances angiogenesis in vitro. The dynamic relationship between these two processes is now evaluated with tunicamycin. Capillary endothelial cells treated with tunicamycin were growth inhibited and could not be reversed with exogenous VEGF(165). Inhibition of angiogenesis is supported by down-regulation of (i) phosphorylated VEGFR1 and VEGFR2 receptors; (ii) VEGF(165)-specific phosphotyrosine kinase activity; and (iii) Matrigel(TM) invasion and chemotaxis. In vivo, tunicamycin prevented the vessel development in Matrigel(TM) implants in athymic Balb/c (nu/nu) mice. Immunohistochemical analysis of CD34 (p < 0.001) and CD144 (p < 0.001) exhibited reduced vascularization. A 3.8-fold increased expression of TSP-1, an endogenous angiogenesis inhibitor in Matrigel(TM) implants correlated with that in tunicamycin (32 h)-treated capillary endothelial cells. Intravenous injection of tunicamycin (0.5 mg/kg to 1.0 mg/kg) per week slowed down a double negative (MDA-MB-435) grade III breast adenocarcinoma growth by ～50-60% in 3 weeks. Histopathological analysis of the paraffin sections indicated significant reduction in vessel size, the microvascular density and tumor mitotic index. Ki-67 and VEGF expression in tumor tissue were also reduced. A significant reduction of N-glycan expression in tumor microvessel was also observed. High expression of GRP-78 in CD144-positive cells supported unfolded protein response-mediated ER stress in tumor microvasculature. ～65% reduction of a triple negative (MDA-MB-231) breast tumor xenograft in 1 week with tunicamycin (0.25 mg/kg) given orally and the absence of systemic and/or organ failure strongly supported tunicamycin's potential for a powerful glycotherapeutic treatment of breast cancer in the clinic.     

The Drosophila CPEB protein Orb2 has a novel expression pattern and is important for asymmetric cell division and nervous system function

Cytoplasmic polyadenylation element binding (CPEB) proteins bind mRNAs to regulate their localization and translation. While the first CPEBs discovered were germline specific, subsequent studies indicate that CPEBs also function in many somatic tissues including the nervous system. Drosophila has two CPEB family members. One of these, orb, plays a key role in the establishment of polarity axes in the developing egg and early embryo, but has no known somatic functions or expression outside of the germline. Here we characterize the other Drosophila CPEB, orb2. Unlike orb, orb2 mRNA and protein are found throughout development in many different somatic tissues. While orb2 mRNA and protein of maternal origin are distributed uniformly in early embryos, this pattern changes as development proceeds and by midembryogenesis the highest levels are found in the CNS and PNS. In the embryonic CNS, Orb2 appears to be concentrated in cell bodies and mostly absent from the longitudinal and commissural axon tracts. In contrast, in the adult brain, the protein is seen in axonal and dendritic terminals. Lethal effects are observed for both RNAi knockdowns and orb2 mutant alleles while surviving adults display locomotion and behavioral defects. We also show that orb2 funtions in asymmetric division of stem cells and precursor cells during the development of the embryonic nervous system and mesoderm.     

Ebolavirus delta-peptide immunoadhesins inhibit marburgvirus and ebolavirus cell entry

With the exception of Reston and Lloviu viruses, filoviruses (marburgviruses, ebolaviruses, and “cuevaviruses”) cause severe viral hemorrhagic fevers in humans. Filoviruses use a class I fusion protein, GP_1,2, to bind to an unknown, but shared, cell surface receptor to initiate virus-cell fusion. In addition to GP_1,2, ebolaviruses and cuevaviruses, but not marburgviruses, express two secreted glycoproteins, soluble GP (sGP) and small soluble GP (ssGP). All three glycoproteins have identical N termini that include the receptor-binding region (RBR) but differ in their C termini. We evaluated the effect of the secreted ebolavirus glycoproteins on marburgvirus and ebolavirus cell entry, using Fc-tagged recombinant proteins. Neither sGP-Fc nor ssGP-Fc bound to filovirus-permissive cells or inhibited GP_1,2-mediated cell entry of pseudotyped retroviruses. Surprisingly, several Fc-tagged Δ-peptides, which are small C-terminal cleavage products of sGP secreted by ebolavirus-infected cells, inhibited entry of retroviruses pseudotyped with Marburg virus GP_1,2, as well as Marburg virus and Ebola virus infection in a dose-dependent manner and at low molarity despite absence of sequence similarity to filovirus RBRs. Fc-tagged Δ-peptides from three ebolaviruses (Ebola virus, Sudan virus, and Taï Forest virus) inhibited GP_1,2-mediated entry and infection of viruses comparably to or better than the Fc-tagged RBRs, whereas the Δ-peptide-Fc of an ebolavirus nonpathogenic for humans (Reston virus) and that of an ebolavirus with lower lethality for humans (Bundibugyo virus) had little effect. These data indicate that Δ-peptides are functional components of ebolavirus proteomes. They join cathepsins and integrins as novel modulators of filovirus cell entry, might play important roles in pathogenesis, and could be exploited for the synthesis of powerful new antivirals.     

Alphacoronavirus transmissible gastroenteritis virus nsp1 protein suppresses protein translation in mammalian cells and in cell-free HeLa cell extracts but not in rabbit reticulocyte lysate

The nsp1 protein of transmissible gastroenteritis virus (TGEV), an alphacoronavirus, efficiently suppressed protein synthesis in mammalian cells. Unlike the nsp1 protein of severe acute respiratory syndrome coronavirus, a betacoronavirus, the TGEV nsp1 protein was unable to bind 40S ribosomal subunits or promote host mRNA degradation. TGEV nsp1 also suppressed protein translation in cell-free HeLa cell extract; however, it did not affect translation in rabbit reticulocyte lysate (RRL). Our data suggested that HeLa cell extracts and cultured host cells, but not RRL, contain a host factor(s) that is essential for TGEV nsp1-induced translational suppression.     

Activation of DNA damage response signaling in lung adenocarcinoma A549 cells following oxygen beam irradiation

Oxygen beams are high linear energy transfer (LET) radiation characterized by higher relative biological effectiveness than low LET radiation. The aim of the current study was to determine the signaling differences between γ- and oxygen ion-irradiation. Activation of various signaling molecules was looked in A549 lung adenocarcinoma cells irradiated with 2Gy oxygen, 2Gy or 6Gy γ-radiation. Oxygen beam was found to be three times more cytotoxic than γ-radiation. By 4h there was efficient repair of DNA in A549 cells exposed to 2Gy or 6Gy gamma radiation but not in cells exposed to 2Gy oxygen beam as determined by γ-H2AX counting. Number of ATM foci was found to be significantly higher in cells exposed to 2Gy oxygen beam. Percentage of cells showing ATR foci were more with gamma however number of foci per cell were more in case of oxygen beam. Oxygen beam irradiated cells showed phosphorylation of Chk1, Chk2 and p53. Many apoptotic nuclei were seen by DAPI staining in cells exposed to oxygen beam. The noteworthy finding of this study is the activation of the sensor proteins, ATM and ATR by oxygen irradiation and the significant activation of Chk1, Chk2 and p53 only in the oxygen beam irradiated cells.     

The inheritance of resistance to Verticillium wilt caused by race 1 isolates of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in the lettuce cultivar La Brillante

Verticillium wilt of lettuce caused by Verticillium dahliae can cause severe economic damage to lettuce producers. Complete resistance to race 1 isolates is available in Lactuca sativa cultivar (cv.) La Brillante and understanding the genetic basis of this resistance will aid development of new resistant cultivars. F₁ and F₂ families from crosses between La Brillante and three iceberg cultivars as well as a recombinant inbred line population derived from L. sativa cv. Salinas 88 × La Brillante were evaluated for disease incidence and disease severity in replicated greenhouse and field experiments. One hundred and six molecular markers were used to generate a genetic map from Salinas 88 × La Brillante and for detection of quantitative trait loci. Segregation was consistent with a single dominant gene of major effect which we are naming Verticillium resistance 1 (Vr1). The gene described large portions of the phenotypic variance (R ² = 0.49–0.68) and was mapped to linkage group 9 coincident with an expressed sequence tag marker (QGD8I16.yg.ab1) that has sequence similarity with the Ve gene that confers resistance to V. dahliae race 1 in tomato. The simple inheritance of resistance indicates that breeding procedures designed for single genes will be applicable for developing resistant cultivars. QGD8I16.yg.ab1 is a good candidate for functional analysis and development of markers suitable for marker-assisted selection.     

In vitro regeneration through organogenesis and somatic embryogenesis in pigeon pea [Cajanus cajan (L.) Millsp.] cv. JKR105

In vitro regeneration of pigeon pea through organogenesis and somatic embryogenesis was demonstrated with pigeon pea cv. JKR105. Embryonic axes explants of pigeon pea showed greater regeneration of shoot buds on 2.5 mg L⁻¹ 6-benzylaminopurine (BAP) in the medium, followed by further elongation at lower concentrations. Rooting of shoots was observed on half-strength Murashige and Skoog (MS) medium with 2 % sucrose and 0.5 mg L⁻¹ 3-indolebutyric acid (IBA). On the other hand, the regeneration of globular embryos from cotyledon explant was faster and greater with thidiazuron (TDZ) than BAP with sucrose as carbohydrate source. These globular embryos were maturated on MS medium with abscisic acid (ABA) and finally germinated on half-strength MS medium at lower concentrations of BAP. Comparison of regeneration pathways in pigeon pea cv. JKR105 showed that the turnover of successful establishment of plants achieved through organogenesis was more compared to somatic embryogenesis, despite the production of more embryos than shoot buds.     

An efficient gel-phase synthesis of peptides on a high capacity flexible crosslinked polystyrene support: comparison with Merrifield resin.

A highly solvating copolymer was prepared in high yield by introducing a flexible crosslinker, 1,4-butanedioldimethacrylate, into the polystyrene matrix by a free radical aqueous suspension polymerization. A 2 mol% crosslinked resin showed rigidity and mechanical characteristics comparable to those of divinylbenzene-crosslinked polystyrene (Merrifield resin, DVB-PS) support. Swelling and solvation characteristics of the new resin, BDDMA-PS, were much higher than DVB-PS support in all solvents used for solid phase peptide synthesis. The diacrylate crosslinks in the resin network were found to be highly stable even after 48 h treatment with neat TFA, 6 N HCl and 6 N KOH at 110 degrees C. To demonstrate the usefulness of the new resin in high capacity peptide synthesis, a typical difficult peptide, acyl carrier protein (ACP) fragment (65-74), was synthesized on commercially available 1 mol% crosslinked DVB-PS and 2 mol% crosslinked BDDMA-PS resins under identical conditions. A protocol using NMP/DMSO mediated coupling was employed for chain assembly. The yield and purity of the product from BDDMA-PS resin was higher than when the DVB-PS resin was used. The mechanistic reason behind the synthetic efficiency of the new resin was found to be its ability to induce random coil conformation to the growing peptide chains.