TLR-induced inflammation in cystic fibrosis and non-cystic fibrosis airway epithelial cells

Cystic fibrosis (CF) is a genetic disease characterized by severe neutrophil-dominated airway inflammation. An important cause of inflammation in CF is Pseudomonas aeruginosa infection. We have evaluated the importance of a number of P. aeruginosa components, namely lipopeptides, LPS, and unmethylated CpG DNA, as proinflammatory stimuli in CF by characterizing the expression and functional activity of their cognate receptors, TLR2/6 or TLR2/1, TLR4, and TLR9, respectively, in a human tracheal epithelial line, CFTE29o(-), which is homozygous for the DeltaF508 CF transmembrane conductance regulator mutation. We also characterized TLR expression and function in a non-CF airway epithelial cell line 16HBE14o(-). Using RT-PCR, we demonstrated TLR mRNA expression. TLR cell surface expression was assessed by fluorescence microscopy. Lipopeptides, LPS, and unmethylated CpG DNA induced IL-8 and IL-6 protein production in a time- and dose-dependent manner. The CF and non-CF cell lines were largely similar in their TLR expression and relative TLR responses. ICAM-1 expression was also up-regulated in CFTE29o(-) cells following stimulation with each agonist. CF bronchoalveolar lavage fluid, which contains LPS, bacterial DNA, and neutrophil elastase (a neutrophil-derived protease that can activate TLR4), up-regulated an NF-kappaB-linked reporter gene and increased IL-8 protein production in CFTE29o(-) cells. This effect was abrogated by expression of dominant-negative versions of MyD88 or Mal, key signal transducers for TLRs, thereby implicating them as potential anti-inflammatory agents for CF.     

Protein-Bound Acrolein

Abstract : Several lines of evidence support the role of oxidative stress, including increased lipid peroxidation, in the pathogenesis of Alzheimer's disease (AD). Lipid peroxidation generates various reactive aldehydes, such as 4-hydroxynonenal (HNE), which have been detected immunochemically in AD, particularly in neurofibrillary tangels, one of the major diagnostic lesions in AD brains. A recent study demonstrated that acrolein, the most reactive among the α, β-unsaturated aldehyde products of lipid peroxidation, could be rapidly incorporated into proteins, generating a carbonyl derivative, a marker of oxidative stress to proteins. The current studies used an antibody raised against acrolein-modified keyhole limpet hemocyanin (KLH) to test whether acrolein modification of proteins occurs in AD. Double immunofluorescence revealed strong acrolein-KLH immunoreactivity in more than half of all paired helical filament (PHF)-1-labeled neurofibrillary tangles in AD cases. Acrolein-KLH immunoreactivity was also evident in a few neurons lacking PHF-1-positive neurofibrillary tangles. Light acrolein-KLH immunoreactivity occurred in dystrophic neurites surrounding the amyloid-β core, which itself lacked acrolein-KLH staining. The pattern of acrolein-KLH immunostaining was similar to that of HNE. Control brains did not contain any acrolein-KLH-immunoreactive structures. The current results suggest that protein-bound acrolein is a powerful marker of oxidative damage to protein and support the hypothesis that lipid peroxidation and oxidative damage to protein may play a crucial role in the formation of neurofibrillary tangles and to neuronal death in AD.     

Infection of soybean and pea nodules by Rhizobium spp. purine auxotrophs in the presence of 5-aminoimidazole-4-carboxamide riboside.

Purine auxotrophs of various Rhizobium species are symbiotically defective, usually unable to initiate or complete the infection process. Earlier studies demonstrated that, in the Rhizobium etli-bean symbiosis, infection by purine auxotrophs is partially restored by supplementation of the plant medium with 5-amino-imidazole-4-carboxamide (AICA) riboside, the unphosphorylated form of the purine biosynthetic intermediate AICAR. The addition of purine to the root environment does not have this effect. In this study, purine auxotrophs of Rhizobium fredii HH303 and Rhizobium leguminosarum 128C56 (bv. viciae) were examined. Nutritional and genetic characterization indicated that each mutant was blocked in purine biosynthesis prior to the production of AICAR. R. fredii HH303 and R. leguminosarum 128C56 appeared to be deficient in AICA riboside transport and/or conversion into AICAR, and the auxotrophs derived from them grew very poorly with AICA riboside as a purine source. All of the auxotrophs elicited poorly developed, uninfected nodules on their appropriate hosts. On peas, addition of AICA riboside or purine to the root environment led to enhanced nodulation; however, infection threads were observed only in the presence of AICA riboside. On soybeans, only AICA riboside was effective in enhancing nodulation and promoting infection. Although AICA riboside supplementation of the auxotrophs led to infection thread development on both hosts, the numbers of bacteria recovered from the nodules were still 2 or more orders of magnitude lower than in fully developed nodules populated by wild-type bacteria. The ability to AICA riboside to promote infection by purine auxotrophs, despite serving as a very poor purine source for these strains, supports the hypothesis that AICAR plays a role in infection other than merely promoting bacterial growth.     

Pak4 induces premature senescence via a pathway requiring p16INK4/p19ARF and mitogen-activated protein kinase signaling

Exposure of primary cells to mitogenic stimuli or oncogenes often causes them to undergo premature senescence. This is most likely a protective function that prevents uncontrolled proliferation. Pak4 is a target for the Rho GTPase Cdc42. Pak4 is overexpressed in human tumor cell lines, and it is the only member of the Pak family that is highly transforming in immortalized fibroblasts. Here we show that in primary fibroblasts, activated Pak4 inhibits cell proliferation and promotes premature senescence. Furthermore, Pak4 expression levels are upregulated in response to stimuli that promote senescence. Pak4-induced arrest appears to be mediated by a pathway that requires the ERK mitogen-activated protein kinase, as well as the cell cycle inhibitors p16^INK4 and p19^ARF. These new results describing a role for Pak4 in senescence are important for understanding why this protein is associated with cancer and how it promotes transformation in immortalized cells.     

The genetic diversity and evolution of field pea (<Emphasis Type="Italic">Pisum</Emphasis>) studied by high throughput retrotransposon based insertion polymorphism (RBIP) marker analysis

Background  

The genetic diversity of crop species is the result of natural selection on the wild progenitor and human intervention by ancient and modern farmers and breeders. The genomes of modern cultivars, old cultivated landraces, ecotypes and wild relatives reflect the effects of these forces and provide insights into germplasm structural diversity, the geographical dimension to species diversity and the process of domestication of wild organisms. This issue is also of great practical importance for crop improvement because wild germplasm represents a rich potential source of useful under-exploited alleles or allele combinations. The aim of the present study was to analyse a major Pisum germplasm collection to gain a broad understanding of the diversity and evolution of Pisum and provide a new rational framework for designing germplasm core collections of the genus.     

The Streamlined Genome of Phytomonas spp. Relative to Human Pathogenic Kinetoplastids Reveals a Parasite Tailored for Plants

Members of the family Trypanosomatidae infect many organisms, including animals, plants and humans. Plant-infecting trypanosomes are grouped under the single genus Phytomonas, failing to reflect the wide biological and pathological diversity of these protists. While some Phytomonas spp. multiply in the latex of plants, or in fruit or seeds without apparent pathogenicity, others colonize the phloem sap and afflict plants of substantial economic value, including the coffee tree, coconut and oil palms. Plant trypanosomes have not been studied extensively at the genome level, a major gap in understanding and controlling pathogenesis. We describe the genome sequences of two plant trypanosomatids, one pathogenic isolate from a Guianan coconut and one non-symptomatic isolate from Euphorbia collected in France. Although these parasites have extremely distinct pathogenic impacts, very few genes are unique to either, with the vast majority of genes shared by both isolates. Significantly, both Phytomonas spp. genomes consist essentially of single copy genes for the bulk of their metabolic enzymes, whereas other trypanosomatids e.g. Leishmania and Trypanosoma possess multiple paralogous genes or families. Indeed, comparison with other trypanosomatid genomes revealed a highly streamlined genome, encoding for a minimized metabolic system while conserving the major pathways, and with retention of a full complement of endomembrane organelles, but with no evidence for functional complexity. Identification of the metabolic genes of Phytomonas provides opportunities for establishing in vitro culturing of these fastidious parasites and new tools for the control of agricultural plant disease.     

Kanamycin resistance as a selectable marker for plastid transformation in tobacco

We report on a novel chimeric gene that confers kanamycin resistance on tobacco plastids. The kan gene from the bacterial transposon Tn5, encoding neomycin phosphotransferase (NPTII), was placed under control of plastid expression signals and cloned between rbcL and ORF512 plastid gene sequences to target the insertion of the chimeric gene into the plastid genome. Transforming plasmid pTNH32 DNA was introduced into tobacco leaves by the biolistic procedure, and plastid transformants were selected by their resistance to 50 g/ml of kanamycin monosulfate. The regenerated plants uniformly transmitted the transplastome to the maternal progeny. Resistant clones resulting from incorporation of the chimeric gene into the nuclear genome were also obtained. However, most of these could be eliminated by screening for resistance to high levels of kanamycin (500 g/ml). Incorporation of kan into the plastid genome led to its amplification to a high copy number, about 10000 per leaf cell, and accumulation of NPTII to about 1% of total cellular protein.     

Nucleotide diversity of vernalization and flowering‐time‐related genes in a germplasm collection of meadow fescue (Festuca pratensis Huds. syn. Lolium pratense (Huds.) Darbysh.)

In plant species, control of flowering time is an important factor for adaptation to local natural environments. The Vrn1, CO, FT1 and CK2α genes are key components in the flowering‐specific signaling pathway of grass species. Meadow fescue is an agronomically important forage grass species, which is naturally distributed across Europe and Western Asia. In this study, meadow fescue flowering‐time‐related genes were resequenced to assess nucleotide diversity in European and Western Asian subpopulations. Identified sequence polymorphisms were then converted into PCR‐based molecular genetic markers, and a meadow fescue germplasm collection was genotyped to investigate global allelic variation. Lower nucleotide diversities were observed for the Vrn1 and CO orthologs, while relatively higher values were observed for the FT1 and casein kinase II α‐subunit (CK2α) orthologs. The nucleotide diversity for FT1 orthologs in the Western Asian subpopulation was significantly higher than those of the European subpopulation. Similarly, significant differences in nucleotide diversity for the remaining genes were observed between several combinations of subpopulation. The global allele distribution pattern was consistent with observed level of nucleotide diversity. These results suggested that the degree of purifying selection acting on the genes differs according to geographical location. As previously shown for model plant species, functional specificities of flowering‐time‐related genes may also vary according to environmental conditions.