Metabolism and translocation of allantoin in ureide-producing grain legumes

Transfer of the nitrogen and carbon of allantoin to amino acids and protein of leaflets, stems and petioles, apices, peduncles, pods, and seeds of detached shoots of nodulated cowpea (Vigna unguiculata L. Walp. cv. Caloona) plants was demonstrated following supply of [2-¹⁴C], [1,3-¹⁵N]allantoin in the transpiration stream. Throughout vegetative and reproductive growth all plant organs showed significant ureolytic activity and readily metabolized [2-¹⁴C]allantoin to ¹⁴CO₂. A metabolic pathway for ureide nitrogen utilization via allantoic acid, urea, and ammonia was indicated. Levels of ureolytic activity in extracts from leaves and roots of nodulated cowpea were consistently maintained at higher levels than in non-nodulated, NO₃⁻ grown plants.

[¹⁴C]Ureides were recovered in extracts of aphids (Aphis craccivora and Macrosiphum euphorbieae) feeding at different sites on cowpea plants supplied with [2-¹⁴C]allantoin through the transpiration stream or to the upper surface of single leaflets. The data indicated that the ureides were effectively transferred from xylem or leaf mesophyll to phloem, and then translocated in phloem to fruits, apices, and roots.

     

Distinct steps of cross-linking, self-association, and maturation of tropoelastin are necessary for elastic fiber formation

Elastic fibers play an important role in the characteristic resilience of many tissues. The assembly of tropoelastin into a fibrillar matrix is a complex stepwise process and the deposition and cross-linking of tropoelastin are believed to be key steps of elastic fiber formation. However, the detailed mechanisms of elastic fiber assembly have not been defined yet. Here, we demonstrate the relationship between deposition and the cross-linking/maturation of tropoelastin. Our data show that a C-terminal half-fragment of tropoelastin encoded by exons 16-36 (BH) is deposited onto microfibrils, yet we detect very limited amounts of the cross-linking amino acid, desmosine, an indicator of maturation, whereas the N-terminal half-fragment encoded by exons 2-15 (FH) was deficient for both deposition and cross-linking, suggesting that elastic fiber formation requires full-length tropoelastin molecules. A series of experiments using mutant BH fragments, lacking either exon 16 or 30, or a deletion of both exons showed that self-association of tropoelastin polypeptides was an early step in elastic fiber assembly. Immunofluorescence and Western blot assay showed that the treatment of cell culture medium or conditioned medium with beta-aminopropionitrile to inhibit cross-linking, prevented both the deposition and polymerization of tropoelastin. In conclusion, our present results support the view that self-association and oxidation by lysyl oxidase precedes tropoelastin deposition onto microfibrils and the entire molecule of tropoelastin is required for this following maturation process.     

Production of high levels of poly‐3‐hydroxybutyrate in plastids of Camelina sativa seeds

Poly‐3‐hydroxybutyrate (PHB) production in plastids of Camelina sativa seeds was investigated by comparing levels of polymer produced upon transformation of plants with five different binary vectors containing combinations of five seed‐specific promoters for expression of transgenes. Genes encoding PHB biosynthetic enzymes were modified at the N‐terminus to encode a plastid targeting signal. PHB levels of up to 15% of the mature seed weight were measured in single sacrificed T₁ seeds with a genetic construct containing the oleosin and glycinin promoters. A more detailed analysis of the PHB production potential of two of the best performing binary vectors in a Camelina line bred for larger seed size yielded lines containing up to 15% polymer in mature T₂ seeds. Transmission electron microscopy showed the presence of distinct granules of PHB in the seeds. PHB production had varying effects on germination, emergence and survival of seedlings. Once true leaves formed, plants grew normally and were able to set seeds. PHB synthesis lowered the total oil but not the protein content of engineered seeds. A change in the oil fatty acid profile was also observed. High molecular weight polymer was produced with weight‐averaged molecular weights varying between 600 000 and 1 500 000, depending on the line. Select lines were advanced to later generations yielding a line with 13.7% PHB in T₄ seeds. The levels of polymer produced in this study are the highest reported to date in a seed and are an important step forward for commercializing an oilseed‐based platform for PHB production.     

Production of polyhydroxybutyrate in switchgrass, a value-added co-product in an important lignocellulosic biomass crop

Polyhydroxyalkanoate bio-based plastics made from renewable resources can reduce petroleum consumption and decrease plastic waste disposal issues as they are inherently biodegradable in soil, compost and marine environments. In this paper, the successful engineering of the biomass crop switchgrass ( Panicum virgatum L.) for the synthesis of polyhydroxybutyrate (PHB) is reported. Polymer production was monitored in more than 400 primary transformants grown under in vitro and glasshouse conditions. Plants containing up to 3.72% dry weight of PHB in leaf tissues and 1.23% dry weight of PHB in whole tillers were obtained. Results from the analysis of the polymer distribution at the cellular and whole plant levels are presented, and target areas for the improvement of PHB production are highlighted. Polymer accumulation was also analysed in the T₁ generation obtained from controlled crosses of transgenic plants. This study presents the first successful expression of a functional multigene pathway in switchgrass, and demonstrates that this high-yielding biomass crop is amenable to the complex metabolic engineering strategies necessary to produce high-value biomaterials with lignocellulose-derived biofuels.     

Identification of a physiological phosphorylation site of the herpes simplex virus 1-encoded protein kinase Us3 which regulates its optimal catalytic activity in vitro and influences its function in infected cells

Us3 is a serine/threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). Here, we report the identification of a physiological Us3 phosphorylation site on serine at position 147 (Ser-147) which regulates its protein kinase activity in vitro. Moreover, mutation of this site influences Us3 function, including correct localization of the enzyme and induction of the usual morphological changes in HSV-1-infected cells. These conclusions are based on the following observations: (i) in in vitro kinase assays, a domain of Us3 containing Ser-147 was specifically phosphorylated by Us3 and protein kinase A, while a mutant domain in which Ser-147 was replaced with alanine was not; (ii) in vitro, alanine replacement of Ser-147 (S147A) in Us3 resulted in significant impairment of the kinase activity of the purified molecule expressed in a baculovirus system; (iii) phosphorylation of Ser-147 in Us3 tagged with the monomeric fluorescent protein (FP) VenusA206K (VenusA206K-Us3) from Vero cells infected with a recombinant HSV-1 encoding VenusA206K-Us3 was specifically detected using an antibody that recognizes phosphorylated serine or threonine residues with arginine at the -3 and -2 positions; and (iv) the S147A mutation influenced some but not all Us3 functions, including the ability of the protein to localize itself properly and to induce wild-type cytopathic effects in infected cells. Our results suggest that some of the regulatory activities of Us3 in infected cells are controlled by phosphorylation at Ser-147.     

Application of a Combination of a Knowledge-Based Algorithm and 2-Stage Screening to Hypothesis-Free Genomic Data on Irinotecan-Treated Patients for Identification of a Candidate Single Nucleotide Polymorphism Related to an Adverse Effect

Interindividual variation in a drug response among patients is known to cause serious problems in medicine. Genomic information has been proposed as the basis for “personalized” health care. The genome-wide association study (GWAS) is a powerful technique for examining single nucleotide polymorphisms (SNPs) and their relationship with drug response variation; however, when using only GWAS, it often happens that no useful SNPs are identified due to multiple testing problems. Therefore, in a previous study, we proposed a combined method consisting of a knowledge-based algorithm, 2 stages of screening, and a permutation test for identifying SNPs. In the present study, we applied this method to a pharmacogenomics study where 109,365 SNPs were genotyped using Illumina Human-1 BeadChip in 168 cancer patients treated with irinotecan chemotherapy. We identified the SNP rs9351963 in potassium voltage-gated channel subfamily KQT member 5 (KCNQ5) as a candidate factor related to incidence of irinotecan-induced diarrhea. The p value for rs9351963 was 3.31×10⁻⁵ in Fisher''s exact test and 0.0289 in the permutation test (when multiple testing problems were corrected). Additionally, rs9351963 was clearly superior to the clinical parameters and the model involving rs9351963 showed sensitivity of 77.8% and specificity of 57.6% in the evaluation by means of logistic regression. Recent studies showed that KCNQ4 and KCNQ5 genes encode members of the M channel expressed in gastrointestinal smooth muscle and suggested that these genes are associated with irritable bowel syndrome and similar peristalsis diseases. These results suggest that rs9351963 in KCNQ5 is a possible predictive factor of incidence of diarrhea in cancer patients treated with irinotecan chemotherapy and for selecting chemotherapy regimens, such as irinotecan alone or a combination of irinotecan with a KCNQ5 opener. Nonetheless, clinical importance of rs9351963 should be further elucidated.     

Drosophila DREF acting via the JNK pathway is required for thorax development

The Drosophila Jun N-terminal kinase (JNK) gene basket (bsk) promoter contains a DNA replication-related element (DRE)-like sequence, raising the possibility of regulation by the DNA replication-related element-binding factor (DREF). Chromatin immunoprecipitation assays with anti-DREF IgG showed the bsk gene promoter region to be effectively amplified. Luciferase transient expression assays revealed the DRE-like sequence to be important for bsk gene promoter activity, and knockdown of DREF decreased the bsk mRNA level and the bsk gene promoter activity. Furthermore, knockdown of DREF in the notum compartment of wing discs by pannier-GAL4 and UAS-DREFIR resulted in a split thorax phenotype. Monitoring of JNK activity in the wing disc by LacZ expression in a puckered (puc)-LacZ enhancer trap line revealed the reduction in DREF knockdown clones. These findings indicate that DREF is involved in regulation of Drosophila thorax development via actions on the JNK pathway.     

Reevaluation of host ranges of feline leukemia virus subgroups

We reevaluated the host ranges of feline leukemia virus (FeLV) subgroups A, B and C using pseudotype assays based on recombinant NB-tropic murine leukemia virus, which is not usually blocked after viral entry in mammalian cells. Pseudotype viruses of FeLV-B and -C infected a variety of cell lines from many mammalian species. Unexpectedly, FeLV-A pseudotype viruses of two independent isolates from the UK and US also infected a variety of non-feline cell lines including cells from humans, rabbits, pigs and minks. Moreover, both isolates of FeLV-A productively infected human embryonic kidney 293 and mink Mv-1-Lu cells. We conclude that FeLV-A is not strictly ecotropic.     

Tyk2-signaling plays an important role in host defense against Escherichia coli through IL-23-induced IL-17 production by gammadelta T cells

Tyrosine kinase 2 (Tyk2), a member of the JAK-signal transducer family, is involved in intracellular signaling triggered by various cytokines, including IL-23. We have recently reported that resident gammadelta T cells in the peritoneal cavity of naive mice produced IL-17 in response to IL-23. In this study, we examined importance of Tyk2-mediated signaling in the IL-17 production by gammadelta T cells using Tyk2 deficient (-/-) mice. Gammadelta T cells in the peritoneal cavity of Tyk2(-/-) mice displayed effecter/memory phenotypes and TCR V repertoire similar to those in Tyk2(+/+) mice and produced comparable level of IL-17 to those in Tyk2(+/+) mice in response to PMA and ionomycin, indicating normal differentiation to IL-17-producing effectors in the absence of Tyk2-signaling. However, gammadelta T cells in Tyk2(-/-) mice produced less amount of IL-17 in response to IL-23 in vitro than those in Tyk2(+/+) mice. Similarly, gammadelta T cells in the peritoneal cavity of Tyk2(-/-) mice showed severely impaired IL-17 production after an i.p. infection with E. coli despite comparable level of IL-23 production to Tyk2(+/+) mice. As a consequence, Tyk2(-/-) mice showed a reduced infiltration of neutrophils and severely impaired bacterial clearance after Escherichia coli infection. These results indicate that Tyk2-signaling is critical for IL-23-induced IL-17 production by gammadelta T cells, which is involved in the first line of host defense by controlling neutrophil-mediated immune responses.     

Comparison of a coq7 deletion mutant with other respiration-defective mutants in fission yeast

Among the steps in ubiquinone biosynthesis, that catalyzed by the product of the clk-1/coq7 gene has received considerable attention because of its relevance to life span in Caenorhabditis elegans. We analyzed the coq7 ortholog (denoted coq7) in Schizosaccharomyces pombe, to determine whether coq7 has specific roles that differ from those of other coq genes. We first confirmed that coq7 is necessary for the penultimate step in ubiquinone biosynthesis, from the observation that the deletion mutant accumulated the ubiquinone precursor demethoxyubiquinone-10 instead of ubiquinone-10. The coq7 mutant displayed phenotypes characteristic of other ubiquinone-deficient Sc. pombe mutants, namely, hypersensitivity to hydrogen peroxide, a requirement for antioxidants for growth on minimal medium, and an elevated production of sulfide. To compare these phenotypes with those of other respiration-deficient mutants, we constructed cytochrome c (cyc1) and coq3 deletion mutants. We also assessed accumulation of oxidative stress in various ubiquinone-deficient strains and in the cyc1 mutant by measuring mRNA levels of stress-inducible genes and the phosphorylation level of the Spc1 MAP kinase. Induction of ctt1, encoding catalase, and apt1, encoding a 25 kDa protein, but not that of gpx1, encoding glutathione peroxidase, was indistinguishable in four ubiquinone-deficient mutants, indicating that the oxidative stress response operates at similar levels in the tested strains. One new phenotype was observed, namely, loss of viability in stationary phase (chronological life span) in both the ubiquinone-deficient mutant and in the cyc1 mutant. Finally, Coq7 was found to localize in mitochondria, consistent with the possibility that ubiquinone biosynthesis occurs in mitochondria in yeasts. In summary, our results indicate that coq7 is required for ubiquinone biosynthesis and the coq7 mutant is not distinguishable from other ubiquinone-deficient mutants, except that its phenotypes are more pronounced than those of the cyc1 mutant.