15N-ammonium and 15N-nitrate uptake of a 15-year-old Picea abies plantation

Throughfall nitrogen of a 15-year-old Picea abies (L.) Karst. (Norway spruce) stand in the Fichtelgebirge, Germany, was labeled with either ¹⁵N-ammonium or ¹⁵N-nitrate and uptake of these two tracers was followed during two successive growing seasons (1991 and 1992). ¹⁵N-labeling (62 mg ¹⁵N m^-2 under conditions of 1.5 g N m^-2 atmospheric nitrogen deposition) did not increase N concentrations in plant tissues. The ¹⁵N recovery within the entire stand (including soils) was 94%±6% of the applied ¹⁵N-ammonium tracer and 100%±6% of the applied ¹⁵N-nitrate tracer during the 1st year of investigation. This decreased to 80%±24% and 83%±20%, respectively, during the 2nd year. After 11 days, the ¹⁵N tracer was detectable in 1-year-old spruce needles and leaves of understory species. After 1 month, tracer was detectable in needle litter fall. At the end of the first growing season, more than 50% of the ¹⁵N taken up by spruce was assimilated in needles, and more than 20% in twigs. The relative distribution of recovered tracer of both ¹⁵N-ammonium and ¹⁵N-nitrate was similar within the different foliage age classes (recent to 11-year-old) and other compartments of the trees. ¹⁵N enrichment generally decreased with increasing tissue age. Roots accounted for up to 20% of the recovered ¹⁵N in spruce; no enrichment could be detected in stem wood. Although ¹⁵N-ammonium and ¹⁵N-nitrate were applied in the same molar quantities (¹⁵NH ₄ ⁺ : ¹⁵NO ₃ ^- =1:1), the tracers were diluted differently in the inorganic soil N pools (¹⁵NH ₄ ⁺ /NH ₄ ⁺ : ¹⁵NO ₃ ^- /NO ₃ ^- =1:9). Therefore the measured ¹⁵N amounts retained by the vegetation do not represent the actual fluxes of ammonium and nitrate in the soil solution. Use of the molar ammonium-to-nitrate ratio of 9:1 in the soil water extract to estimate ¹⁵N uptake from inorganic N pools resulted in a 2–4 times higher ammonium than nitrate uptake by P. abies.     

Targeting the interleukin-15/interleukin-15 receptor system in inflammatory autoimmune diseases

Interleukin (IL)-15 is a dangerous inflammatory cytokine that induces tumor-necrosis factor-α, IL-1β and inflammatory chemokines. It inhibits self-tolerance mediated by IL-2 mediated activation-induced cell death and facilitates maintenance of CD8⁺ memory T-cell survival including that of self-directed memory cells. Disordered IL-15 expression has been reported in patients with an array of inflammatory autoimmune diseases. A series of therapeutic agents that inhibit IL-15 action have been introduced, including the soluble IL-15 receptor (IL-15R) α chain, mutant IL-15, and antibodies directed against the IL-15 cytokine and against the IL-2R/IL-15R β subunit used by IL-2 and IL-15.     

Precursor role of 15alpha-hydroxyestradiol and 15alpha-hydroxyandrostenedione in the formation of estetrol

Studies were designed to elucidate the origin of estetrol (15alpha-hydroxyestriol (estra-1,3,5(10)triene-3,15alpha,17beta-tetrol) or E4) during late human pregnancy. 3H-Labelled 15alpha-hydroxyestradiol (3,15alpha-dihydroxyestra-1,3,5(10)-trien-17-one or 15E2) and 14C-labelled 17beta-estradiol (estra-1,3,5(10)-triene-3,17beta-diol or E2) were infused into the fetus during transfusion in utero for erythroblastosis fetalis, and in another study the same substrates were injected intravenously into the maternal circulation. In a third study, 3H-labelled 15alpha-hydroxyandrostenedion (15alpha-hydroxyandrost-4-ene-3,17-dione or 15delta4) and 14C-labelled E2 were infused into the fetus. Maternal urine was collected for 5--6 days, and after Glusulase hydrolysis, the following metabolites were isolated: estriol (estra-1,3,5(10)-triene-3,16alpha,17beta-triol or E3) containing 14C only and 15alpha-hydroxyestrone (3,15alpha-dihydroxyestra-1,3,5(10)-trien-17-one or 15E1), 15E2, and E4, all containing both labels. From the isotope content of these metabolites, it was concluded that E4 was derived from both fetal E2 and 15delta4 and only partially via 15E2. When administered to the fetus E2 and 15delta4 contributed approximately equal amounts to urinary E4. The yield of 15alpha-hydroxylated estrogens from E2 injected into the mother was very low indicating the predominantly fetal origin of the 15alpha-hydroxylase. 15delta4 was a better precursor than E2 for urinary 15E2.     

Combined IL-15/IL-15Ralpha immunotherapy maximizes IL-15 activity in vivo

IL-15 has substantial potential as an immunotherapeutic agent for augmenting immune responses. However, the activity of IL-15 is mediated by a unique mechanism in which the cytokine is transpresented by cell-bound high-affinity IL-15Ralpha to target cells expressing the IL-15Rbeta and the common gamma-chain. Thus, the efficacy of administered IL-15 alone may be limited by the availability of free IL-15Ralpha. We now show that administration of soluble IL-15/IL-15Ralpha complexes greatly enhanced IL-15 half-life and bioavailability in vivo. Treatment of mice with this complex, but not with IL-15 alone, resulted in robust proliferation of memory CD8 T cells, NK cells, and NK T cells. The activity of the complex required IL-15Rbeta, but not IL-15Ralpha, expression by the responding cells and was IL-7-independent. Interestingly, IL-15/IL-15Ralpha immunotherapy also caused naive CD8 T cell activation and development into effector cells and long-term memory T cells. Lastly, complexed IL-15, as compared with IL-15 alone, dramatically reduced tumor burden in a model of B16 melanoma. These findings hold significant importance for the use of IL-15 as a potential adjuvant/therapeutic and inducer of homeostatic proliferation, without the necessity for prior immunodepletion.     

Semisynthesis of Arg15, Glu15, Met15, and Nle15-aprotinin involving enzymatic peptide bond resynthesis

The semisynthesis of homologues of aprotinin, the bovine pancreatic trypsin inhibitor, is described. The P₁ lysine¹⁵ residue was replaced by two methods. The first procedure, which consisted of two enzymatic steps for the incorporation of other amino acids has previously been described. The second approach consisted of six steps of both enzymatic and chemical nature. The modified inhibitor, in which the lysine¹⁵-alanine¹⁶ peptide bond is hydrolyzed, was used as the starting material. All carboxyl groups of the modified inhibitor were esterified with methanol; the lysine¹⁵ methylester group was then selectively hydrolyzed. Afterward, lysine¹⁵ itself was split off. Arginine, glutamic acid, methionine, andl-2-aminohexanoic acid (norleucine, Nle) were incorporated using water-soluble carbodiimide combined with an acylation catalyst. The methylester group was used to prevent polymerization. The reactive-site peptide bonds were resynthesized using either chymotrypsin or trypsin.     

N15: the linear phage-plasmid

The lambdoid phage N15 of Escherichia coli is very unusual among temperate phages in that its prophage is not integrated into chromosome but is a linear plasmid molecule with covalently closed ends. Upon infection the phage DNA circularises via cohesive ends, then phage-encoded enzyme, protelomerase, cuts at an inverted repeat site and forms hairpin ends (telomeres) of the linear plasmid prophage. Replication of the N15 prophage is initiated at an internally located ori site and proceeds bidirectionally resulting in formation of duplicated telomeres. Then the N15 protelomerase cuts duplicated telomeres generating two linear plasmid molecules with hairpin telomeres. Stable inheritance of the plasmid prophage is ensured by partitioning operon similar to the F factor sop operon. Unlike F sop, the N15 centromere consists of four inverted repeats dispersed in the genome. The multiplicity and dispersion of centromeres are required for efficient partitioning of a linear plasmid. The centromeres are located in N15 genome regions involved in phage replication and control of lysogeny, and binding of partition proteins at these sites regulates these processes. Two N15-related lambdoid Siphoviridae phages, φKO2 in Klebsiella oxytoca and pY54 in Yersinia enterocolitica, also lysogenize their hosts as linear plasmids, as well as Myoviridae marine phages VP882 and VP58.5 in Vibrio parahaemolyticus and ΦHAP-1 in Halomonas aquamarina. The genomes of all these phages contain similar protelomerase genes, lysogeny modules and replication genes, as well as plasmid-partitioning genes, suggesting that these phages may belong to a group diverged from a common ancestor.     

Roles of the 15-kDa Selenoprotein (Sep15) in Redox Homeostasis and Cataract Development Revealed by the Analysis of Sep 15 Knockout Mice

The 15-kDa selenoprotein (Sep15) is a thioredoxin-like, endoplasmic reticulum-resident protein involved in the quality control of glycoprotein folding through its interaction with UDP-glucose:glycoprotein glucosyltransferase. Expression of Sep15 is regulated by dietary selenium and the unfolded protein response, but its specific function is not known. In this study, we developed and characterized Sep15 KO mice by targeted removal of exon 2 of the Sep15 gene coding for the cysteine-rich UDP-glucose:glycoprotein glucosyltransferase-binding domain. These KO mice synthesized a mutant mRNA, but the shortened protein product could be detected neither in tissues nor in Sep15 KO embryonic fibroblasts. Sep15 KO mice were viable and fertile, showed normal brain morphology, and did not activate endoplasmic reticulum stress pathways. However, parameters of oxidative stress were elevated in the livers of these mice. We found that Sep15 mRNA was enriched during lens development. Further phenotypic characterization of Sep15 KO mice revealed a prominent nuclear cataract that developed at an early age. These cataracts did not appear to be associated with severe oxidative stress or glucose dysregulation. We suggest that the cataracts resulted from an improper folding status of lens proteins caused by Sep15 deficiency.     

Proteomic analysis of differentially expressed proteins between Xiangyou 15 variety and the mutant M15

A high oleic acid rapeseed material MI5 （derived from Xiangyou 15 variety） has been received more attention for its significant effect for human health. And it has almost the same physiological characteristic with Xiangyou 15 variety. To find out the difference between high oleic acid rapeseed material and Xiangyou 15 seedling, a comparative proteomic approach based on 2-DE and mass spectrometry was adopted. A total of 277 protein spots showed a significant change in intensity by more than 2.0-fold from M15 compared with Xiangyou 15 variety. Among them, 48 spots that changed at least 3.0-fold were excised from gels and successfully identified by MALDI-TOF/TOF MS. The identified proteins involved in metabolism of carbohydrate and energy （75%）, stress and defense （8.3%）, photosynthesis （6.3%）, protein metabolism （2.1%） and other functions （8.3%）. Then real-time quantitative PCR （qPCR） analysis was used to verify the expression levels of differentially expressed proteins, but the results did well agree with the proteomic results. In this work, most of the proteins involved in metabolism of carbohydrate and energy have higher expression in M15, which may reveal M15 has higher metabolism ability. These results provided much information to understand the differences between high oleic acid rapeseed material and Xiangyou 15 variety, which will be useful to screen high oleic rapeseed materials in seedling period.     

Properties of the soluble arachidonic acid 15-lipoxygenase and 15-hydroperoxide isomerase from the oomycete Saprolegnia parasitica

The soluble hydroperoxide isomerase and 15-lipoxygenase activities were partially purified from the oomycete Saprolegnia parasitica and some of their properties characterized. Both enzymes co-eluted with a molecular weight of 145,000-150,000 on Sephacryl S-300 chromatography. The enzyme activities also co-eluted on DEAE Sephadex ion exchange chromatography and hydroxylapatite chromatography. Both activities showed similar responses to pH and temperature. Both enzymes showed parallel inhibition by p-hydroxymercuribenzoate and eicosatetraynoic acid. The partially purified hydroperoxide isomerase showed an apparent km of 166 microM and a Vmax of 5.3 mumol/min/mg protein for exogenous 15-HPETE. It was not stimulated by calcium. These results suggest that the soluble hydroperoxide isomerase and 15-lipoxygenase activities from S. parasitica are both contained on the same protein or protein complex.     

Regulation of T-lymphocyte mitogenesis by the leukocyte product 15-hydroxy-eicosatetraenoic acid (15-HETE)

Synthesis of lipoxygenase metabolites of [¹⁴C]arachidonic acid by mouse spleen lymphocyte cultures was inhibited by the leukocyte product 15-hydroxy-eicosatetraenoic acid (15-HETE) in a dose-dependent manner. In parallel experiments, the influence of 15-HETE on mitogenesis in spleen lymphocyte cultures was examined. 15-HETE at concentrations similar to those which inhibited cellular lipoxygenases progressively inhibited mitogenesis induced by the T-cell mitogen PHA but had no significant effect on the mitogenic response to the B-cell mitogen LPS. The inhibitory response was maximal when 15-HETE was added within 8 hr of exposure to PHA. Several analogs of 15-HETE having progressively fewer double bonds were tested in the same systems. 15-OH,20:3 had approximately the same potency as 15-HETE in inhibiting both mitogenesis and formation of metabolites from [¹⁴C]arachidonic acid. 15-OH, 20:2 and 15-OH,20:0 were much less active in either assay. Mitogenesis, induced in spleen cell cultures by the tumor promoter phorbol myristate acetate, was also blocked by 15-HETE. These experiments indicate that lipoxygenase metabolites of arachidonic acid may play an important role in T-lymphocyte blastogenesis and suggest that 15-HETE, via its ability to selectively inhibit cellular lipoxygenases, may function as an endogenous regulator of T-lymphocyte responses.     

Structural studies of the capsular polysaccharide from Streptococcus pneumoniae types 15B and 15C

The structures of the capsular polysaccharides (S-15B and S-15C) from Streptococcus pneumoniae types 15B and 15C have been investigated by using n.m.r. spectroscopy, methylation analysis, and various specific degradations. It is concluded that the polysaccharides are composed of pentasaccharide repeating-units having the following structure: (Formula: see text). In this structure, R is H (80%) or CH2CH2N+Me3 (20%). S-15B further contains O-acetyl groups, approximately 0.7 per repeating unit, which have not been located. The capsular polysaccharides S-15F and S-15A, which have been studied previously, are also composed of pentasaccharide repeating-units, containing the same sequence of sugars, but in a linear arrangement.     

Differential nucleocytoplasmic trafficking between the related endocytic proteins Eps15 and Eps15R.

Eps15 and Eps15R are constitutive components of clathrin-coated pits that are required for clathrin-dependent endocytosis. The most striking difference between these two related proteins is that Eps15R is also found in the nucleus, whereas Eps15 is excluded from this compartment at steady state. To better understand the individual functions of these two proteins, the mechanisms responsible for their different localization were investigated. Interestingly, some mutants of Eps15 were found in the nucleus. This nuclear localization was correlated with the loss of the last approximately 100 amino acids of Eps15, suggesting the presence of a nuclear export signal (NES) within this region. As expected, the last 25 amino acids contain a leucine-rich sequence matching with classical NESs, show a leptomycin B-sensitive nuclear export activity, and bind to the exportin CRM1 in a leucine residue-dependent manner. In contrast, no NES could be found in Eps15R, a result in keeping with its constitutive nuclear localization that appears to be regulated by alternative splicing. Altogether, these results are the first characterization of nucleocytoplasmic shuttling signals for endocytic proteins. They also provide an explanation for the different nuclear localization of Eps15 and Eps15R and further evidence for a possible nuclear function for Eps15 protein family members.     

The Prader-Willi syndrome and 15-15 translocation]

The association of the Willi-Prader syndrome and a t(15q15q) is reported. This, in conjunction with an earlier report of this association, suggests that a gene related to the Willi-Prader syndrome may be present on chromosome 15.     

15 Combinatorial chemistry in the prebiotic environment

The pathway leading to the origin of life presumably included a process by which polymers were synthesized abiotically from simpler compounds on the early Earth, then encapsulated to form protocells. Previous studies have reported that mineral surfaces can concentrate and organize activated mononucleotides, thereby promoting their polymerization into RNA-like molecules. However, a plausible prebiotic activation mechanism has not been established, and minerals cannot form cellular compartments. We are exploring ways in which nonactivated mononucleotides can undergo polymerization and encapsulation. We found that small yields of RNA-like molecules are synthesized by a condensation reaction when mixtures of amphiphilic lipids and mononucleotides are exposed to cycles of dehydration and rehydration. The lipids concentrate and organize the monomers within multilamellar liquid-crystalline matrices that self-assemble in the dry state. The chemical potential driving the polymerization reaction is supplied by the anhydrous conditions in which water becomes a leaving group, with heat providing activation energy. Significantly, the polymeric products are encapsulated in trillions of microscopic compartments upon rehydration. Each compartment is unique in its composition and contents, and can be considered to be an experiment in a natural version of combinatorial chemistry that would be ubiquitous in the prebiotic environment. A successful experiment would be a compartment that captured polymers capable of catalyzing their own replication. If this can be reproduced in the laboratory, it would represent a significant step toward understanding the origin of cellular life.