Paeonol promoted 2-aminofluorene and p-aminobenzoic acid acetylations by mononuclear leucocytes from Sprague-Dawley rats

Following exposure of rats to the arylamine carcinogen 2-aminofluorene, DNA-carcinogen adducts were found in the target tissues of the liver and bladder, and also in circulating leucocytes. This work investigated how paeonol affects arylamine (2-aminofluorene and p-aminobenzoic acid) acetylations in rat leucocytes. Evidence is presented showing that rat mononuclear leucocytes are capable of acetylating 2-aminofluorene and p-aminobenzoic acid. Paeonol promoted 2-aminofluorene and p-aminobenzoic acid acetylation. Cultured lymphocytes produced about twice as much N-acetyl-2-aminofluorene from 2-aminofluorene and 2.2-fold as much N-acetyl-p-aminobenzoic acid from p-aminobenzoic acid as monocytes. After cotreatment with paeonol, the lymphocyte and monocyte cultures indicated that paeonol did increase 2-aminofluorene and p-aminobenzoic acid acetylations.     

Evasion of host defenses by measles virus: wild-type measles virus infection interferes with induction of Alpha/Beta interferon production

Measles is a highly contagious disease currently responsible for over one million childhood deaths, particularly in the developing world. Since alpha/beta interferons (IFNs) are pivotal players both in nonspecific antiviral immunity and in specific cellular responses, their induction or suppression by measles virus (MV) could influence the outcome of a viral infection. In this study we compare the IFN induction and sensitivity of laboratory-passaged attenuated MV strains Edmonston and Moraten with those of recent wild-type viruses isolated and passaged solely on human peripheral blood mononuclear cells (PBMC) or on the B958 marmoset B-cell line. We report that two PBMC-grown wild-type measles isolates and two B958-grown strains of MV induce 10- to 80-fold-lower production of IFN by phytohemagglutinin-stimulated peripheral blood lymphocytes (PBL) compared to Edmonston and Moraten strains of measles. Preinfection of PBL with these non-IFN-inducing MV isolates prevents Edmonston-induced but not double-stranded-RNA-induced IFN production. This suggests that the wild-type viruses can actively inhibit Edmonston-induced IFN synthesis and that this is not occurring by double-stranded RNA. Furthermore, the wild-type MV is more sensitive than Edmonston MV to the effect of IFN. MV is thus able to suppress the synthesis of the earliest mediator of antiviral immunity, IFN-alpha/beta. This could have important implications in the virulence and spread of MV.     

Asynchronous accumulation of lettuce infectious yellows virus RNAs 1 and 2 and identification of an RNA 1 trans enhancer of RNA 2 accumulation

Time course and mutational analyses were used to examine the accumulation in protoplasts of progeny RNAs of the bipartite Crinivirus, Lettuce infectious yellow virus (LIYV; family Closteroviridae). Hybridization analyses showed that simultaneous inoculation of LIYV RNAs 1 and 2 resulted in asynchronous accumulation of progeny LIYV RNAs. LIYV RNA 1 progeny genomic and subgenomic RNAs could be detected in protoplasts as early as 12 h postinoculation (p.i.) and accumulated to high levels by 24 h p.i. The LIYV RNA 1 open reading frame 2 (ORF 2) subgenomic RNA was the most abundant of all LIYV RNAs detected. In contrast, RNA 2 progeny were not readily detected until ca. 36 h p.i. Mutational analyses showed that in-frame stop codons introduced into five of seven RNA 2 ORFs did not affect accumulation of progeny LIYV RNA 1 or RNA 2, confirming that RNA 2 does not encode proteins necessary for LIYV RNA replication. Mutational analyses also supported that LIYV RNA 1 encodes proteins necessary for replication of LIYV RNAs 1 and 2. A mutation introduced into the LIYV RNA 1 region encoding the overlapping ORF 1B and ORF 2 was lethal. However, mutations introduced into only LIYV RNA 1 ORF 2 resulted in accumulation of progeny RNA 1 near or equal to wild-type RNA 1. In contrast, the RNA 1 ORF 2 mutants did not efficiently support the trans accumulation of LIYV RNA 2. Three distinct RNA 1 ORF 2 mutants were analyzed and all exhibited a similar phenotype for progeny LIYV RNA accumulation. These data suggest that the LIYV RNA 1 ORF 2 encodes a trans enhancer for RNA 2 accumulation.     

Enhancement of in vitro growth of papaya multishoots by aeration

Efficient micropropagation of papaya (Carica papaya L.) has become crucial for multiplication of specific sex types of papaya or transgenic lines resistant to virus infection. In this study, aeration at different intervals with a 0.02 μm filter disc in the closure of culture flasks ensured exchange of gas components. The effect of aeration on development of multibuds to multishoots was investigated. Multibuds grown in culture flasks after one-week without aeration followed by a two-week aeration treatment caused a 41% increase in the number of shoots ≥0.5 cm, 42% increase in leaf expansion, and 17% increase in leaf numbers in comparison with unaerated materials. Ethylene and oxygen concentrations in the culture flasks were measured by gas chromatography and oxygen electrode at weekly intervals during the culture period. Oxygen concentrations were slightly different between aerated and unaerated culture flasks. Ethylene in the unaerated flask reached the highest level (0.11 ppm) 2 weeks after the treatment, while accumulation of ethylene in the aerated flasks was not detected. The multishoots grown for 3 weeks without aeration showed growth retardation on leaves and epinasty on petioles. This revised version was published online in June 2006 with corrections to the Cover Date.     

Synthesis and the biological evaluation of arylnaphthalene lignans as anti-hepatitis B virus agents

We have previously shown that helioxanthin can suppress human hepatitis B virus gene expression. A series of helioxanthin analogues were synthesized and evaluated for their anti-hepatitis B virus activity. Modifications at the lactone rings and methylenedioxy unit of helioxanthin can modulate the antiviral activity. Among them, compound 32 is the most effective anti-HBV agent. Compound 32 can suppress the secretion of viral surface antigen and e antigen in HepA2 cells with EC₅₀ values of 0.06 and 0.14 μM, respectively. Compound 32 not only inhibited HBV DNA with wild-type and lamivudine-resistant strain but also suppressed HBV mRNA, core protein and viral promoters. In this study, a full account of the preparation, structure–activity relationships of helioxanthin analogues, and the possible mechanism of anti-HBV activity of this class of compounds are presented. This type of compounds possesses unique mode of action differing from existing therapeutic drugs. They are potentially new anti-HBV agents.     

Real-time molecular methods to detect infectious viruses

Waterborne transmitted viruses pose a public health threat due to their stability in aquatic environment and the easy transmission with high morbidity rates at low infectious doses. Two major challenge of virus analysis include a lack of adequate information in infectivity and the inability to cultivate certain epidemiologically important viruses in vitro. The use of fluorescent probes in conjunction with fluorescence microscopy allows us to reveal dynamic interactions of the viruses with different cellular structures in living cells that are impossible to detect by immunological or PCR-based experiments. Real-time viral detection in vivo provides sufficient information regarding multiple steps in infection process at molecular level, which will be valuable for the prevention and control of viral infection.     

Chromosomal integration of a synthetic expression control sequence achieves poly‐γ‐glutamate production in a Bacillus subtilis strain

Poly‐γ‐glutamate (γ‐PGA) has applications in food, medical, cosmetic, animal feed, and wastewater industries. Bacillus subtilis DB430, which possesses the γ‐PGA synthesis ywsC‐ywtAB genes in its chromosome, cannot produce γ‐PGA. An efficient synthetic expression control sequence (SECS) was introduced into the upstream region of the ywtABC genes, and this resulted in γ‐PGA‐producing B. subtilis mutant strains. Mutant B. subtilis PGA6‐2 stably produces high levels of γ‐PGA in medium A without supplementation of extra glutamic acid or ammonium chloride. The mutant B. subtilis PGA 6‐2 is not only a γ‐PGA producer, but it is also a candidate for the genetic and metabolic engineering of γ‐PGA production. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Nutritional Energy Stimulates NAD+ Production to Promote Tankyrase-Mediated PARsylation in Insulinoma Cells

The poly-ADP-ribosylation (PARsylation) activity of tankyrase (TNKS) regulates diverse physiological processes including energy metabolism and wnt/β-catenin signaling. This TNKS activity uses NAD⁺ as a co-substrate to post-translationally modify various acceptor proteins including TNKS itself. PARsylation by TNKS often tags the acceptors for ubiquitination and proteasomal degradation. Whether this TNKS activity is regulated by physiological changes in NAD⁺ levels or, more broadly, in cellular energy charge has not been investigated. Because the NAD⁺ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) in vitro is robustly potentiated by ATP, we hypothesized that nutritional energy might stimulate cellular NAMPT to produce NAD⁺ and thereby augment TNKS catalysis. Using insulin-secreting cells as a model, we showed that glucose indeed stimulates the autoPARsylation of TNKS and consequently its turnover by the ubiquitin-proteasomal system. This glucose effect on TNKS is mediated primarily by NAD⁺ since it is mirrored by the NAD⁺ precursor nicotinamide mononucleotide (NMN), and is blunted by the NAMPT inhibitor FK866. The TNKS-destabilizing effect of glucose is shared by other metabolic fuels including pyruvate and amino acids. NAD⁺ flux analysis showed that glucose and nutrients, by increasing ATP, stimulate NAMPT-mediated NAD⁺ production to expand NAD⁺ stores. Collectively our data uncover a metabolic pathway whereby nutritional energy augments NAD⁺ production to drive the PARsylating activity of TNKS, leading to autoPARsylation-dependent degradation of the TNKS protein. The modulation of TNKS catalytic activity and protein abundance by cellular energy charge could potentially impose a nutritional control on the many processes that TNKS regulates through PARsylation. More broadly, the stimulation of NAD⁺ production by ATP suggests that nutritional energy may enhance the functions of other NAD⁺-driven enzymes including sirtuins.