Effects of recombinant cholera toxin B subunit on IL-1beta production by macrophages in vitro

Recombinant cholera toxin B subunit (rCTB) is a safe and potent mucosal adjuvant. As a clue to the mechanism of the adjuvant effect of rCTB, the profile of cytokines secreted in vitro by the mouse peritoneal macrophage (Mphi) treated with rCTB was examined. IL-1beta secretion, intracellular production, and expression of its mRNA of LPS-stimulated Mphi was greatly enhanced by treatment with rCTB. IL-1beta production in response to other microbial stimulators, such as Pansorbin, Sansorbin, insoluble peptidoglycan, and Taxol, was also potentiated by rCTB. Mphi pretreated with rCTB before 24 hr could maintain the ability to produce a high level of IL-1beta, suggesting that this ability may be involved in the adjuvant activity of rCTB on Mphi stimulation. The possibility of close association between rCTB and signal transduction of a Toll-like receptor family in Mphi is discussed.     

Role of internalization in the pathogenicity of Shiga toxin-producing Escherichia coli infection in a gnotobiotic murine model

We investigated the role of bacterial internalization in the killing caused by Shiga toxin-producing Escherichia coli (STEC) infection using a gnotobiotic murine model. A high number of internalized STEC was found in the colonic epithelial cells of STEC-infected mice by both an ex vivo assay and transmission electron microscopy. Most of these mice were killed within 10 days after infection. However, the implantation of lactic acid bacteria in such mice before infection markedly decreased the number of internalized STECs and also completely protected these hosts from killing by a STEC infection. The inhibition of such internalization by immunoglobulin also prevented the hosts from being killed. The Shiga toxin levels in these hosts indicated an inhibition of the penetration of Shiga toxins produced in the colon to the underlying tissue. These results suggested that the internalization plays an important role in the pathogenicity caused by STEC infection in a gnotobiotic murine model.     

Iron-catalyzed reaction products of alpha-tocopherol with 1-palmitoyl-2-linoleoyl-3-sn-phosphatidylcholine (13S)-hydroperoxide

Alpha-tocopherol was reacted with 1-palmitoyl-2-[(9Z,11E)-(S)-13-hydroperoxy-9,11-octadecadienoyl]-3-sn-phosphatidylcholine (13-PLPC-OOH) in the presence of a lipid-soluble iron chelate, Fe(III) acetylacetonate, in methanol at 37 degrees C. The reaction product was isolated and identified as a mixture of 1-palmitoyl-2-[(10E)-(12S,13S)-9-(8a-dioxy-alpha-tocopherone)-12,13-epoxy-10-octadecenoyl]-3-sn-phosphatidylcholine and 1-palmitoyl-2-[(9Z)-(12S,13S)-11-(8a-dioxy-alpha-tocopherone)-12,13-epoxy-9-octadecenoyl]-3-sn-phosphatidylcholine (TOO-epoxyPLPC), in which the 12,13-epoxyperoxyl radicals derived from 13-PLPC-OOH attacked the 8a-position of the alpha-tocopheroxyl radical. The iron and ascorbate-catalyzed reaction of 13-PLPC-OOH with alpha-tocopherol in phosphatidylcholine (PC) liposomes was assessed by measuring the reaction products of alpha-tocopherol. When 13-PLPC-OOH and alpha-tocopherol were added in saturated dimyristoyl-PC liposomes, the products were TOO-epoxyPLPC, alpha-tocopherylquinone, and epoxy-alpha-tocopherylquinones. In 1-palmitoyl-2-linoleoyl-PC (PLPC) liposomes, alpha-tocopherol could react with both the 13-PLPC-OOH derived 12,13-epoxyperoxyl radicals and the PLPC-derived peroxyl radicals and formed the addition products together with alpha-tocopherylquinone and epoxy-alpha-tocopherylquinones. Therefore, the iron-catalyzed decomposition of phospholipid hydroperoxides primarily produces epoxyperoxyl radicals, which react with the 8a-carbon centered radical of alpha-tocopherol in liposomal systems.     

Characterization of the rRNA locus of Pfiesteria piscicida and development of standard and quantitative PCR-based detection assays targeted to the nontranscribed spacer

Pfiesteria piscicida is a heterotrophic dinoflagellate widely distributed along the middle Atlantic shore of the United States and associated with fish kills in the Neuse River (North Carolina) and the Chesapeake Bay (Maryland and Virginia). We constructed a genomic DNA library from clonally cultured P. piscicida and characterized the nontranscribed spacer (NTS), small subunit, internal transcribed spacer 1 (ITS1), 5.8S region, ITS2, and large subunit of the rRNA gene cluster. Based on the P. piscicida ribosomal DNA sequence, we developed a PCR-based detection assay that targets the NTS. The assay specificity was assessed by testing clonal P. piscicida and Pfiesteria shumwayae, 35 additional dinoflagellate species, and algal prey (Rhodomonas sp.). Only P. piscicida and nine presumptive P. piscicida isolates tested positive. All PCR-positive products yielded identical sequences for P. piscicida, suggesting that the PCR-based assay is species specific. The assay can detect a single P. piscicida zoospore in 1 ml of water, 10 resting cysts in 1 g of sediment, or 10 fg of P. piscicida DNA in 1 micro g of heterologous DNA. An internal standard for the PCR assay was constructed to identify potential false-negative results in testing of environmental sediment and water samples and as a competitor for the development of a quantitative competitive PCR assay format. The specificities of both qualitative and quantitative PCR assay formats were validated with >200 environmental samples, and the assays provide simple, rapid, and accurate methods for the assessment of P. piscicida in water and sediments.     

Glial uptake of neurotransmitter glutamate from the extracellular fluid studied in vivo by microdialysis and (13)C NMR

Glial uptake of neurotransmitter glutamate (GLU) from the extracellular fluid was studied in vivo in rat brain by (13)C NMR and microdialysis combined with gas-chromatography/mass-spectrometry. Brain GLU C5 was (13)C enriched by intravenous [2,5-(13)C]glucose infusion, followed by [(12)C]glucose infusion to chase (13)C from the small glial GLU pool. This leaves [5-(13)C]GLU mainly in the large neuronal metabolic pool and the vesicular neurotransmitter pool. During the chase, the (13)C enrichment of whole-brain GLU C5 was significantly lower than that of extracellular GLU (GLU(ECF)) derived from exocytosis of vesicular GLU. Glial uptake of neurotransmitter [5-(13)C]GLU(ECF) was monitored in vivo through the formation of [5-(13)C,(15)N]GLN during (15)NH(4)Ac infusion. From the rate of [5-(13)C,(15)N]GLN synthesis (1.7 +/- 0.03 micromol/g/h), the mean (13)C enrichment of extracellular GLU (0.304 +/- 0.011) and the (15)N enrichment of precursor NH(3) (0.87 +/- 0.014), the rate of synthesis of GLN (V'(GLN)), derived from neurotransmitter GLU(ECF), was determined to be 6.4 +/- 0.44 micromol/g/h. Comparison with V(GLN) measured previously by an independent method showed that the neurotransmitter provides 80-90% of the substrate GLU pool for GLN synthesis. Hence, under our experimental conditions, the rate of 6.4 +/- 0.44 micromol/g/h also represents a reasonable estimate for the rate of glial uptake of GLU(ECF), a process that is crucial for protecting the brain from GLU excitotoxicity.     

Elevated incidence of loss of heterozygosity (LOH) in an sgs1 mutant of Saccharomyces cerevisiae: roles of yeast RecQ helicase in suppression of aneuploidy,interchromosomal rearrangement,and the simultaneous incidence of both events during mitotic growth

The SGS1 gene of Saccharomyces cerevisiae is a member of the RecQ helicase family, which includes the human BLM, WRN and RECQL4 genes responsible for Bloom and Werner's syndrome and Rothmund-Thomson syndrome, respectively. Cells defective in any of these genes exhibit a higher incidence of genome instability. We previously demonstrated that various genetic alterations were detectable as events leading to loss of heterozygosity (LOH) in S. cerevisiae diploid cells, utilizing a hemizygous URA3 marker placed at the center of the right arm of chromosome III. Analyses of chromosome structure in LOH clones by pulse field gel electrophoresis (PFGE) and PCR, coupled with a genetic method, allow identification of genetic alterations leading to the LOH. Such alterations include chromosome loss, chromosomal rearrangements at various locations and intragenic mutation. In this work, we have investigated the LOH events occurring in cells lacking the SGS1 gene. The frequencies of all types of LOH events, excluding intragenic mutation, were increased in sgs1 null mutants as compared to the wild-type cells. Loss of chromosome III and chromosomal rearrangements were increased 13- and 17-fold, respectively. Further classification of the chromosomal rearrangements confirmed that two kinds of events were especially increased in the sgs1 mutants: (1) ectopic recombination between chromosomes, that is, unequal crossing over and translocation (46-fold); and (2) allelic crossing over associated with chromosome loss (40-fold). These findings raise the possibility that the Sgs1 protein is involved in the processing of recombination intermediates as well as in the prevention of recombination repair during chromosome DNA replication. On the other hand, intrachromosomal deletions between MAT and HMR were increased only slightly (2.9-fold) in the sgs1 mutants. These results clearly indicate that defects in the SGS1 gene function lead to an elevated incidence of LOH in multiple ways, including chromosome loss and interchromosomal rearrangements, but not intrachromosomal deletion.     

Identification of human Kir2.2 (KCNJ12) gene encoding functional inward rectifier potassium channel in both mammalian cells and Xenopus oocytes

Arginine residue at position 285 (R285) in the intracellular C-terminal domain of inward rectifier potassium channel Kir2.2 is conserved in many species, but missing in previously reported human Kir2.2 sequences. We here identified the human Kir2.2 gene in normal individuals, which contained R285 in the deduced amino-acid sequence (hKir2.2/R285). All 30 individuals we examined were homozygous for Kir2.2/R285 gene. The hKir2.2/R285 was electrophysiologically functional in both mammalian cells and Xenopus oocytes. However, the hKir2.2 missing R285 was functional only in Xenopus oocytes, but not in mammalian cells. Thus, R285 in Kir2.2 is important for its functional expression in mammalian cells.