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.     

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.     

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.     

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.     

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.     

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.     

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.     

Comparative Studies on the Soluble Components of Adenovirus Types 9 and 15 and the Intermediate Strain 9-15

Five different soluble components of adenovirus types 9, 9-15, and 15 have been identified. These are: (i) a slowly sedimenting, trypsin-resistant, incomplete hemagglutinin (HA). (This component was demonstrable by hemagglutination-enhancement (HE) tests in the presence of heterotypic antisera against members of Rosen's subgroups II and III, but not of subgroup I); (ii) a slowly sedimenting, trypsin-resistant, complete HA, causing only a partial agglutination of cells; (iii) a rapidly sedimenting, incomplete HA, demonstrable by HE tests in the presence of heterotypic antisera against members of all Rosen's subgroups. (Trypsin treatment of this component caused a conversion into slowly sedimenting incomplete HA); (iv) a group-specific complement-fixing (CF) antigen devoid of HA activity; and (v) a rapidly sedimenting, trypsin-sensitive, complete HA, which in the electron microscope was found to represent a dodecahedral aggregate of 12 pentons (a dodecon). On the basis of their biological and physicochemical characteristics, the first four components were interpreted to represent (i) fibers, (ii) a polymer of a few, probably two, fibers, (iii) pentons, and (iv) hexons, respectively. The length of fibers extending from dodecons and virions was estimated to be 11 to 14 nm. A similar value was suggested from exclusion chromatography experiments. Adenovirus types 9 and 15 fibers were recovered in a position intermediate to that of fibers of types 3 and 4, the lengths of which are 10 and 17 nm, respectively. The sequence of elution of different components of types 9 and 9-15 from an anion exchanger was fibers, fiber-aggregate, pentons, hexons, and dodecons. Type 15 components appeared in the same order except for the fact that dodecons eluted before hexons. The molarities of NaCl required to elute the different types 9 and 9-15 components, excluding hexons, were identical. They were distinctly different from those of the corresponding type 15 components. However, hexons of all three serotypes eluted in proximity to each other and there was a slight tendency for type 9-15 hexons to take a position intermediate to those of types 9 and 15.     

The proregion of mouse BMP15 regulates the cooperative interactions of BMP15 and GDF9

Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) are secreted by the mammalian oocyte and are essential for ovarian follicular development, ovulation, and fertility. However, the secreted forms of the BMP15 and GDF9 proteins and the nature of cooperative molecular interactions between BMP15 and GDF9 previously reported have not been fully characterized. In this study, we found that recombinant mouse BMP15 and GDF9 are secreted as cleaved mature and proregion proteins, with BMP15 also secreted as uncleaved promature protein. Noncovalent interactions were identified between the mature and proregion proteins of each growth factor. Moreover, GDF9 mature protein was found to coimmunoprecipitate with the BMP15 proregion, suggestive of a heteromeric association between BMP15 and GDF9. Mouse GDF9 was found to exist mostly as a dimer of mature protein, in both the presence and absence of BMP15. In contrast, BMP15 formed mostly multimers of proregion and mature protein when combined with GDF9, providing further evidence for heteromeric interaction. Mouse BMP15 was found to act cooperatively with GDF9 in a rat granulosa cell thymidine incorporation bioassay and to signal through the BMPR2 and ACVR1B/TGFBR1/ACVR1C receptor-mediated pathways. Immunoneutralization experiments using GDF9 mature protein antibody indicated that these cooperative interactions are species specific. Additionally, immunoneutralization with proregion antibodies highlighted the involvement of the BMP15 proregion in BMP15/GDF9 cooperative interactions. Taken together, these findings support a novel hypothesis where the extracellular cooperative interactions of recombinant mouse BMP15 and GDF9 are multimeric, involving the proregion of BMP15, and may well be species specific.     

Structural and functional comparison of 15S- and 15R-specific cyclooxygenases from the coral Plexaura homomalla.

It has been known for 30 years that the gorgonian coral Plexaura homomalla contains either 15S- or 15R-configuration prostaglandins (PGs), depending on its location in the Caribbean. Recently we showed that the 15R-PGs in the R-variety of P. homomalla are formed by a unique cyclooxygenase (COX) with 15R oxygenation specificity [Valmsen, K., J?rving, I., Boeglin, W.E., Varvas, K., Koljak, R., Pehk, T., Brash, A.R. & Samel, N. (2001) Proc. Natl. Acad. Sci. USA98, 7700]. Here we describe the cloning and characterization of a closely related COX protein (97% amino acid sequence identity) from the S-variety of P. homomalla. Functional expression of the S-variant COX cDNA in Sf9 insect cells followed by incubation with exogenous arachidonic acid resulted in formation of PG products with > 98% 15S-configuration. Mutational analysis was performed on a suggested active site determinant of C-15 oxygenation specificity, position 349 (Val in all S-specific COX, Ile in 15R-COX). The 15S-COX Val349 to Ile mutant formed 35% 15R-PGs, while the reverse mutation in the 15R-COX (Ile349Val) led to formation of 70% 15S-products. This establishes position 349 as an important determinant of the product stereochemistry at C-15. Our characterization of the enzyme variants demonstrates that very minor sequence divergence accounts for the content of epimeric PGs in the two variants of P. homomalla and that the differences do not arise by isomerization of the products.     

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.     

Formation of 15alpha-hydroxyestriol and 15alpha-hydroxyestradiol from C19 15alpha-hydroxylated precursors in human pregnancy.

A mixture of 3H-15alpha-hydroxyandrostenedione and 14C-15alpha-hydroxydehydroisoandrosterone was injected intravenously into two subjects in the third trimester of pregnancy and, in a second study, directly into two fetuses in utero during transfusion for erythroblastosis fetalis. The urine was collected for 4-5 days and steroid conjugates in the urine were hydrolyzed into sulfate and glucosiduronate fractions. From the glucosiduronate fraction 15alpha-hydroxyestriol, 15alpha-hydroxyestradiol, 15alpha-hydroxyandrostenedione and 15alpha-hydroxydehydroisoandrosterone were isolated. No metabolites were identified in the sulfate fraction of the urine. A marked difference was observed in the metabolism of 15alpha-hydroxyandrostenedione and 15alpha-hydroxydehydroisoandrosterone which is dependent on the route of administration of the substrates. Both substrates were converted to 15alpha-hydroxyestriol and 15alpha-hydroxyestradiol, and the 3H/14C ratios and percentage conversions suggest that 15alpha-hydroxyandrostenedione seems to be a better precursor of the urinary 15alpha-hydroxylated estrogens than 15alpha-hydroxydehydroisoandrosterone. The 3H/14C ratios also suggest that 15alpha-hydroxydehydroisoandrosterone was converted to 15alpha-hydroxyestriol via 15alpha-hydroxyandrostenedione, and that the formation of 15alpha-hydroxyestradiol from 15alpha-hydroxydehydroisoandrosterone via 15alpha-hydroxyandrostenedione is a pathway of minor importance. Finally, 15alpha-hydroxydehydroisoandrosterone was recovered from the urine only when the precursors were injected into the maternal circulation. Also, an unknown metabolite containing only 14C was detected in the glucosiduronate fraction of the urine of each subject.     

Nitrosylation of ISG15 prevents the disulfide bond-mediated dimerization of ISG15 and contributes to effective ISGylation

The expression of the ubiquitin-like molecule ISG15 (UCRP) and protein modification by ISG15 (ISGylation) are strongly activated by interferon, genotoxic stress, and pathogen infection, suggesting that ISG15 plays an important role in innate immune responses. Inducible nitric-oxide synthase (iNOS) is induced by the similar stimuli as ISG15 and enhances the production of nitric oxide (NO), a pleiotropic free radical with antipathogen activity. Here, we report that cysteine residues (Cys-76 and -143 in mouse, Cys-78 in human) of ISG15 can be modified by NO, and the NO modification of ISG15 decreases the dimerization of ISG15. The mutation of the cysteine residue of ISG15 to serine improves total ISGylation. The NO synthase inhibitor S-ethylisothiourea reduces endogenous ISGylation. Furthermore, ectopic expression of iNOS enhanced total ISGylation. Together, these results suggest that nitrosylation of ISG15 enhances target protein ISGylation. This is the first report of a relationship between ISGylation and nitrosylation.     

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.     

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 k_m of 166 μM and a V_max of 5.3 μmol/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.     

Cutting edge: transpresentation of IL-15 by bone marrow-derived cells necessitates expression of IL-15 and IL-15R alpha by the same cells

IL-15 is critical for generation of multiple lymphoid subsets. Recent data have demonstrated a unique aspect of responses to IL-15, in that cells bearing the IL-15Ralpha chain can bind soluble IL-15 and "transpresent" the cytokine to other cells, allowing the latter to respond to IL-15. However, it is unclear whether IL-15 is normally secreted and then becomes bound to surface IL-15Ralpha on bystander cells, or whether transpresentation is mediated by the same cells which synthesize IL-15. Using mixed bone marrow chimeric mice, we present evidence for the latter model, showing that development of NK cells and memory phenotype CD8 T cells necessitates that both IL-15 and IL-15Ralpha be expressed by the same population of cells. These data argue that soluble forms of IL-15 are irrelevant for physiological responses to this cytokine, and the implications of this finding are discussed.     

A comparative analysis of the molecular characteristics of the Arabidopsis CoA pyrophosphohydrolases AtNUDX11, 15, and 15a

Coenzyme A (CoA) is an essential, ubiquitous cofactor in all biological systems, where it acts as the major acyl group carrier in various central metabolic reactions. Although much is known about CoA biosynthesis, it is unclear how the CoA pool is regulated the various cellular compartments. It has been found that the nucleoside diphosphates linked to some moiety X (Nudix) hydrolases, AtNUDX11 and 15, have pyrophosphohydrolase activity toward CoA and its derivatives. In this study we identified two alternatively spliced variants, AtNUDX15 and 15a, produced from the AtNUDX15 gene, and carried out comparative studies of the gene regulation, the kinetic parameters, and the intracellular localization of AtNUDX11, 15, and 15a. The present findings indicate that AtNUDX11 and AtNUDX15(a) function in the hydrolysis of malonyl-CoA in cytosol and succinyl-CoA in the mitochondria, respectively, suggesting their impact not only on CoA biosynthesis but also on various CoA-related pathways such as the TCA cycle.     

Progesterone regulation of preimplantation conceptus growth and galectin 15 (LGALS15) in the ovine uterus

Peri-implantation conceptus (embryo/fetus and associated extraembryonic membranes) growth and development are primarily regulated by secretions from the uterus. This study investigated the effects of progesterone on preimplantation conceptus development and endometrial galectin 15 (LGALS15). Ewes received daily injections of either corn oil (CO) vehicle or 25 mg progesterone (P4) from 36 h postmating to hysterectomy. Treatment with P4 increased blastocyst diameter by 220% on Day 9 and advanced time of elongation of blastocysts to a filamentous conceptus on Day 12. Effects of P4 treatment on blastocyst development were blocked by administration of RU486, a progesterone receptor antagonist. Consistent with early elongation of blastocysts, interferon tau (IFNT) protein was about 50-fold greater in uterine flushes from Day 12 in ewes receiving P4 compared with those receiving CO. Expression of cathepsin L (CTSL) and radical S-adenosyl methionine domain containing 2 (RSAD2), both IFNT-stimulated genes, was increased in endometria of Day 12 P4-treated ewes. LGALS15 mRNA, expressed only in the endometrial luminal epithelium and superficial glands, was detected between Days 9 and 12 and was more abundant in ewes receiving P4 than in those receiving CO on both Days 9 and 12. RU486 treatment ablated P4 induction of LGALS15 mRNA in the endometrial epithelia. LGALS15 protein in uterine flushings was not different on Day 9 but tended to be greater in P4-treated ewes than in those receiving CO on Day 12. The advanced development of blastocysts in P4-treated ewes is hypothesized to involve early induction of specific genes in the endometrial epithelia, such as LGALS15, and undoubtedly components of uterine histotroph.