Accumulation of cytoplasmic beta-catenin and nuclear glycogen synthase kinase 3beta in Epstein-Barr virus-infected cells

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with cancers in immunocompromised populations. EBV establishes a latent infection and immortalizes and transforms B lymphocytes. Several latent proteins have profound effects on cellular growth, including activation of NF-kappaB, phosphatidylinositol 3'-OH kinase (PI3K) signaling, and notch signaling. Activation of PI3K can affect the activity of beta-catenin, the target of the wnt signaling pathway. Deregulation of beta-catenin is associated with a number of malignancies. To determine if beta-catenin is regulated by EBV infection, EBV-infected cells were examined for beta-catenin levels and localization. beta-Catenin was increased in EBV-positive tumor cell lines compared to EBV-negative lines, in EBV-infected Burkitt's lymphoma cell lines, and in EBV-transformed lymphoblastoid cell lines (LCL). In contrast to wnt signaling, EBV consistently induced the accumulation of beta-catenin in the cytoplasm but not the nucleus. The beta-catenin regulating kinase, glycogen synthase kinase 3beta (GSK3beta), was shown to be phosphorylated and inactivated in EBV-infected lymphocytes. Inactivated GSK3beta was localized to the nucleus of EBV-infected LCL. Neither the cytoplasmic accumulation of beta-catenin nor the nuclear inactivation of GSK3beta was affected by the inhibition of PI3K signaling. These data indicate that latent infection with EBV has unique effects on beta-catenin signaling that are distinct from activation of wnt and independent of its effects on PI3K.     

Biosynthesis of pectic galactan by membrane-bound galactosyltransferase from soybean (<Emphasis Type="Italic">Glycine max</Emphasis> Merr.) seedlings

We investigated the properties of a galactosyltransferase (GalT) that is involved in the synthesis of -(14)-galactan side chains of pectins. A membrane preparation of etiolated 6-day-old soybean (Glycine max Merr.) hypocotyls transferred [¹⁴C]Gal from UDP-[¹⁴C]Gal into intact and partially hydrolyzed lupin -(14)-galactans of various chain lengths as exogenous acceptors, while activity to endogenous acceptors was negligible. Maximal activity occurred at pH 6.5 and 20–25°C in the presence of 25 mM Mn²⁺ and 0.75% Triton X-100. The transfer reaction onto the unmodified commercial pectic galactan (M _r>150,000) from lupin we used was very low but increased when the M _r of the galactan was reduced by partial acid hydrolysis. Among the partially hydrolyzed galactans, high-M _r (average M _r 60,000) -(14)-galactan was a more efficient acceptor [specific activity 2,000–3,000 pmol min^–1 (mg protein)^–1] than low-M _r (average M _r 10,000 and 5,000) polymers. Digestion of the radiolabeled product from high-M _r galactan with endo--(14)-galactanase released mainly radioactive -(14)-galactobiose and Gal, indicating that the transfer of [¹⁴C]Gal occurred through -(14)-linkages. HPLC analysis showed that the enzyme also catalyzes incorporation of Gal into pyridylaminated (PA) -(14)-galactooligomers with degree of polymerization at least 5. Gal₇-PA chains were elongated by attachment of one, two, or three Gal residues leading to the formation of Gal_8–10-PA.Abbreviations AGP Arabinogalactan-protein - Ara Arabinose - DP Degree of polymerization - GalA Galacturonic acid - Gal _n -PA Pyridylaminated -(14)-galactooligosaccharides - GalT Galactosyltransferase - MALDI–TOF–MS Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry - Rha Rhamnose Sugars described in this paper belong to the d-series unless otherwise noted     

Enhancement of insulin sensitivity in adipocytes by ginger

Antidiabetic and hypoglycemic drugs have been reported to enhance adipocyte differentiation of 3T3-L1 preadipocytes. We previously reported that ginseng (active constituents: ginsenosides) enhanced the differentiation [1]. In this experiment, effect of some ginger group food extracts on the adipocyte differentiation was investigated using cultured mouse 3T3-L1 preadipocytes. 3T3-L1 cells were grown as monolayer cultures at 37 degrees C in DMEM supplemented by 10% FBS under the atmosphere of 5% CO(2)-95% air. Ginger extracts were found to enhance the adipocyte differentiation. Active constituent was purified and identified as gingerol. In the gingerol-treated cells, insulin-sensitive glucose uptake was increased. It is expected that ginger enhance the insulin-sensitivity, and improve chronic disease, such as diabetes.     

An Epstein-Barr virus protein interacts with Notch

The Epstein-Barr virus (EBV) BamHI A mRNAs were originally identified in cDNA libraries from nasopharyngeal carcinoma, where they are expressed at high levels. The RNAs are differentially spliced to form several open reading frames and also contain the BARF0 open reading frame at the 3' end. One cDNA, RK-BARF0, included a potential endoplasmic reticulum-targeting signal peptide sequence. The RK-BARF0 protein is shown here to interact with the Notch4 ligand binding domain, using yeast two-hybrid screening, coimmunoprecipitation, and confocal microscopy. This interaction induces translocation of a portion of the full-length unprocessed Notch4 to the nucleus by using the Notch nuclear localization signal. These effects of RK-BARF0 on Notch intracellular location indicate that EBV possibly modulates Notch signaling. Unprocessed Notch4 was also detected in immunoprecipitated complexes from EBV-infected cells by using a rabbit antiserum raised against a BARF0-specific peptide. This finding provides additional evidence for expression of RK-BARF0 and its interaction with Notch during EBV infection. In EBV-infected, EBNA2-negative cells, RK-BARF0 induced the expression of EBV latent membrane protein 1 (LMP1), and this induction was dependent on the RK-BARF0/Notch interaction domain. The activation of LMP1 expression by RK-BARF0 may be responsible for expression of LMP1 in EBV latent infections in the absence of EBNA2.     

Three Arabidopsis genes encoding proteins with differential activities for cysteine synthase and beta-cyanoalanine synthase

Three cDNA clones encoding putative cysteine synthases (O-acetylserine (thiol) lyase, EC 4.2.99.8) were isolated from Arabidopsis thaliana and designated AtcysC1, AtcysD1 and AtcysD2, respectively. Southern blot analyses suggested that the corresponding genes were present as a single copy, or at most two copies, in the A. thaliana genome. Escherichia coli complementation analyses confirmed that the cDNAs encode cysteine synthase and the corresponding proteins produced in E. coli clearly showed cysteine synthase activity. In addition, AtcysC1 protein showed beta-cyanoalanine synthase (EC 4.4.1.9) activity, but the other two did not. Kinetic analysis suggests that AtcysC1 actually functions as beta-cyanoalanine synthase rather than cysteine synthase in vivo. The mRNA accumulation of AtcysC1, AtcysD1 and AtcysD2 differed in various organs, but did not change markedly when A. thaliana seedlings were subjected to various stresses, including nutrient deprivation. In vivo targeting experiments indicated that AtcysD1 and AtcysD2 are cytoplasmic isozymes, and AtcysC1 is a mitochondrial isozyme.     

Nematicidal alkaloids and related compounds produced by the fungus Penicillium cf. simplicissimum

A new nematicidal alkaloid, peniprequinolone (1), together with the known alkaloids penigequinolones A and B (2a, 2b), 3-methoxy-4-hydroxy-4-(4'-methoxyphenyl)quinolinone (3), and 3-methoxy-4,6-dihydroxy-4-(4'-methoxyphenyl)quinolinone (4), were isolated from Penicillium cf. simplicissimum (Oudemans) Thom. Cyclopenin (5) and a compound (6a/6b) structurally related to cyclopenin also were isolated from the fungus, and their structures were established by spectroscopic analysis. The biological activities of 1, 2, 3, 4, and 5 were examined by a bioassay with root-lesion nematodes.