Three cadinene derivatives and a prostaglandin-like acid from Chromolaena species

An investigation of the aerial parts of Chromolaena chasleae afforded a prostaglandin-like acid, whose structure followed from the ¹H NMR spectral data and from the reaction with periodate, which afforded a hemiacetal. From a new Chromolaena species, three novel cadinene derivatives were isolated, which were closely related to those isolated previously from this genus.     

Auxin triggers transient local signaling for cell specification in Arabidopsis embryogenesis

The Arabidopsis embryonic root meristem is initiated by the specification of a single cell, the hypophysis. This event critically requires the antagonistic auxin response regulators MONOPTEROS and BODENLOS, but their mechanism of action is unknown. We show that these proteins interact and transiently act in a small subdomain of the proembryo adjacent to the future hypophysis. Here they promote transport of auxin, which then elicits a second response in the hypophysis itself. Our results suggest that hypophysis specification is not the direct result of a primary auxin response but rather depends on cell-to-cell signaling triggered by auxin in adjacent cells.     

IRES-mediated translation of utrophin A is enhanced by glucocorticoid treatment in skeletal muscle cells

Glucocorticoids are currently the only drug treatment recognized to benefit Duchenne muscular dystrophy (DMD) patients. The nature of the mechanisms underlying the beneficial effects remains incompletely understood but may involve an increase in the expression of utrophin. Here, we show that treatment of myotubes with 6alpha-methylprednisolone-21 sodium succinate (PDN) results in enhanced expression of utrophin A without concomitant increases in mRNA levels thereby suggesting that translational regulation contributes to the increase. In agreement with this, we show that PDN treatment of cells transfected with monocistronic reporter constructs harbouring the utrophin A 5'UTR, causes an increase in reporter protein expression while leaving levels of reporter mRNAs unchanged. Using bicistronic reporter assays, we further demonstrate that PDN enhances activity of an Internal Ribosome Entry Site (IRES) located within the utrophin A 5'UTR. Analysis of polysomes demonstrate that PDN causes an overall reduction in polysome-associated mRNAs indicating that global translation rates are depressed under these conditions. Importantly, PDN causes an increase in the polysome association of endogenous utrophin A mRNAs and reporter mRNAs harbouring the utrophin A 5'UTR. Additional experiments identified a distinct region within the utrophin A 5'UTR that contains the inducible IRES activity. Together, these studies demonstrate that a translational regulatory mechanism involving increased IRES activation mediates, at least partially, the enhanced expression of utrophin A in muscle cells treated with glucocorticoids. Targeting the utrophin A IRES may thus offer an important and novel therapeutic avenue for developing drugs appropriate for DMD patients.     

Design and synthesis of a new [18F]fluoropyridine-based haloacetamide reagent for the labeling of oligonucleotides: 2-bromo-N-[3-(2-[18F]fluoropyridin-3-yloxy)propyl]acetamide

Based on the recently highlighted potential of nucleophilic heteroaromatic ortho-radiofluorinations in the preparation of fluorine-18-labeled radiotracers and radiopharmaceuticals for PET, a [(18)F]fluoropyridine-based bromoacetamide reagent has been prepared and used in prosthetic group introduction for the labeling of oligonucleotides. [(18)F]FPyBrA (2-bromo-N-[3-(2-[(18)F]fluoropyridin-3-yloxy)propyl]acetamide) was designed as a radiochemically feasible reagent, its pyridinyl moiety both carrying the radioactive halogen (fluorine-18) and allowing its efficient incorporation via a nucleophilic heteroaromatic substitution, and its 2-bromoacetamide function, ensuring the efficient alkylation of a phosphorothioate monoester group born at the 3'- or 5'-end of single-stranded oligonucleotides. [(18)F]FPyBrA (HPLC-purified) was efficiently prepared in 18-20% non-decay-corrected yield (based on starting [(18)F]fluoride) using a three-step radiochemical pathway in 80-85 min. The developed procedure involves (1) a high-yield nucleophilic heteroaromatic ortho-radiofluorination as the fluorine-18 incorporation-step (70-85% radiochemical yield) and uses [3-(3-tert-butoxycarbonylaminopropoxy)pyridin-2-yl]trimethylammonium trifluoromethanesulfonate as precursor for labeling, followed by (2) rapid and quantitative TFA-removal of the N-Boc-protective group and (3) condensation with 2-bromoacetyl bromide (45-65% radiochemical yield). Typically, 3.3-3.7 GBq (90-100 mCi) of HPLC-purified [(18)F]FPyBrA could be obtained in 80-85 min, starting from 18.5 GBq (500 mCi) of a cyclotron production batch of [(18)F]fluoride. [(18)F]FPyBrA was regioselectively conjugated with 9-mer and 18-mer single-stranded oligonucleotides, provided with a phosphorothioate monoester group at their 3'-end. Both natural phosphodiester DNAs and in vivo-stable 2'-methoxy and -fluoro-modified RNAs were used. Conjugation uses optimized, short-time reaction conditions (MeOH/0.1 M PBS pH 7.4, 15 min, 120 degrees C), both compatible with the chemical stability of the oligonucleotides (ONs) and the half-life of fluorine-18. Conjugated [(18)F]ONs were finally purified by RP-HPLC and desalted using a Sephadex NAP-10 column. The whole radiosynthetic procedure, including the preparation of the fluorine-18-labeled reagent, the conjugation with the oligonucleotide, and the HPLC purification and formulation lasted 140-160 min. [(18)F]FPyBrA represents a valuable alternative to the already reported N-(4-[(18)F]fluorobenzyl)-2-bromoacetamide for the design and development of oligonucleotide-based radiopharmaceuticals for PET.     

NMR-validated structural model for oxidized<Emphasis Type="Italic"> Rhodopseudomonas palustris</Emphasis> cytochrome<Emphasis Type="Italic"> c</Emphasis><Subscript>556</Subscript>

The structure of oxidized Rhodopseudomonas palustris cytochrome c ₅₅₆ has been modeled after that of high-spin cytochrome c from the same bacterium, the latter being the protein with the greatest sequence identity (35%) among all sequenced proteins in the genomes. The two proteins differ in the number of ligands to iron and in spin state, the former being six-coordinate low-spin and the latter five-coordinate high-spin. In order to validate this modeled structure, several structural restraints were obtained by performing a restricted set of NMR experiments, without performing a complete assignment of the protein signals. The aim was to exploit the special restraints arising from the paramagnetism of the metal ion. A total of 43 residual-dipolar-coupling and 74 pseudocontact-shift restraints, which together sampled all regions of the protein, were used in conjunction with over 40 routinely obtained NOE distance restraints. A calculation procedure was undertaken combining the program MODELLER and the solution structure determination program PARAMAGNETIC DYANA, which includes paramagnetism-based restraints. The directions and magnitude of the magnetic susceptibility anisotropy tensor were also calculated. The approach readily provides useful results, especially for paramagnetic metalloproteins of moderate to large dimensions.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-003-0511-2     

Functional genomics of C190T single nucleotide polymorphism in human N-acetyltransferase 2

N-acetyltransferase 2 (NAT2) catalyzes N-acetylation and O-acetylation of many drugs and environmental carcinogens. Genetic polymorphisms in the NAT2 gene have been associated with differential susceptibility to cancers and drug toxicity from these compounds. Single nucleotide polymorphisms (SNPs) have been identified in the human NAT2 coding region. A new allele, NAT2*19, possessing the C190T (R64W) exchange, was recently identified. In order to understand the effect of this new SNP, recombinant NAT2*4 (reference) and NAT2*19 were expressed in yeast (Schizosaccharomyces pombe). The C190T (R64W) SNP in NAT2*19 caused substantial reduction in the NAT2 protein level and stability, but did not cause significant reduction in transformation efficiency or mRNA level. The enzymatic activities for N-acetylation of two arylamine carcinogens (2-aminofluorene, 4-aminobiphenyl), and a sulfonamide drug (sulfamethazine) were over 100-fold lower for NAT2 19 compared to reference NAT2 4. Kinetic studies showed a reduction in Vmax but no significant change in substrate Km. In addition, the SNP caused significant reduction in the O-acetylation of the N-hydroxy-2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine. These results show that NAT2*19 possessing the C190T (R64W) SNP encodes a slow acetylator phenotype for both N- and O-acetylation, due to a reduction in the amount and stability of the NAT2 19 allozyme.     

Sipaucins A-C,sesquiterpenoids from Siparuna pauciflora

The phytochemical investigation of the leaves of Siparuna pauciflora yielded three novel sesquiterpenoids: the germacrane sipaucin A, the elemane sipaucin B and sipaucin C, comprising a new type of carbon skeleton. In addition, four known aporphine alkaloids-nor-boldine, boldine, laurotetanine, and N-methyl-laurotetanine-were obtained. The evaluation of the antiplasmodial activity of the isolated compounds against two strains of Plasmodium falciparum (PoW, Dd2) showed a moderate activity of nor-boldine.