2'-Fluoroarabino- and arabinonucleic acid show different conformations, resulting in deviating RNA affinities and processing of their heteroduplexes with RNA by RNase H

2'-Deoxy-2'-fluoro-arabinonucleic acid (FANA) and arabinonucleic acid (ANA) paired to RNA are substrates of RNase H. The conformation of the natural DNA/RNA hybrid substrates appears to be neither A-form nor B-form. Consistent with this, the conformations of FANA and ANA were found to be intermediate between the A- and B-forms. However, FANA opposite RNA is preferred by RNase H over ANA, and the RNA affinity of FANA considerably exceeds that of ANA. By investigating the conformational boundaries of FANA and ANA residues in crystal structures of A- and B-form DNA duplexes at atomic resolution, we demonstrate that FANA and ANA display subtle conformational differences. The structural data provide insight into the structural requirements at the catalytic site of RNase H. They also allow conclusions with regard to the relative importance of stereoelectronic effects and hydration as modulators of RNA affinity.     

Recognition of O6-benzyl-2′-deoxyguanosine by a perimidinone-derived synthetic nucleoside: a DNA interstrand stacking interaction

The 2′-deoxynucleoside containing the synthetic base 1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-1H-perimidin-2(3H)-one] (dPer) recognizes in DNA the O⁶-benzyl-2′-deoxyguanosine nucleoside (O⁶-Bn-dG), formed by exposure to N-benzylmethylnitrosamine. Herein, we show how dPer distinguishes between O⁶-Bn-dG and dG in DNA. The structure of the modified Dickerson–Drew dodecamer (DDD) in which guanine at position G⁴ has been replaced by O⁶-Bn-dG and cytosine C⁹ has been replaced with dPer to form the modified O⁶-Bn-dG:dPer (DDD-XY) duplex [5′-d(C¹G²C³X⁴A⁵A⁶T⁷T⁸Y⁹G¹⁰C¹¹G¹²)-3′]₂ (X = O⁶-Bn-dG, Y = dPer) reveals that dPer intercalates into the duplex and adopts the syn conformation about the glycosyl bond. This provides a binding pocket that allows the benzyl group of O⁶-Bn-dG to intercalate between Per and thymine of the 3′-neighbor A:T base pair. Nuclear magnetic resonance data suggest that a similar intercalative recognition mechanism applies in this sequence in solution. However, in solution, the benzyl ring of O⁶-Bn-dG undergoes rotation on the nuclear magnetic resonance time scale. In contrast, the structure of the modified DDD in which cytosine at position C⁹ is replaced with dPer to form the dG:dPer (DDD-GY) [5′-d(C¹G²C³G⁴A⁵A⁶T⁷T⁸Y⁹G¹⁰C¹¹G¹²)-3′]₂ duplex (Y = dPer) reveals that dPer adopts the anti conformation about the glycosyl bond and forms a less stable wobble pairing interaction with guanine.     

Entropy versus aromaticity in the conformational dynamics of aromatic rings

Comparison of the results of Car-Parrinello molecular dynamics simulations of isolated benzene, pyrimidine and 1,2,4-triazine molecules reveals that the unusually low population of planar geometry of the benzene ring is caused by entropy effects despite its high aromaticity. The decrease in symmetry of the molecule results in smaller changes in entropy and Gibbs free energy due to out-of-plane deformations of the ring, leading to an increase in the population of planar geometry of the ring. This leads to differences in the topology of potential energy and Gibbs free energy surfaces.

Chain length specificity for activation of cPLA2�� by C1P: use of the dodecane delivery system to determine lipid-specific effects

Previously, our laboratory demonstrated that ceramide-1-phosphate (C1P) specifically activated group IVA cytosolic phospholipase A₂ (cPLA₂α) in vitro. In this study, we investigated the chain length specificity of this interaction. C1P with an acyl-chain of ≥6 carbons efficiently activated cPLA₂α in vitro, whereas C₂-C1P, was unable to do so. Delivery of C1P to cells via the newly characterized ethanol/dodecane system demonstrated a lipid-specific activation of cPLA₂α, AA release, and PGE₂ synthesis (EC₅₀ = 400 nM) when compared to structurally similar lipids. C1P delivered as vesicles in water also induced a lipid-specific increase in AA release. Mass spectrometric analysis demonstrated that C1P delivered via ethanol/dodecane induced a 3-fold increase in endogenous C1P with little metabolism to ceramide. C1P was also more efficiently delivered (>3-fold) to internal membranes by ethanol/dodecane as compared to vesiculated C1P. Using this now established delivery method for lipids, C₂-C1P was shown to be ineffective in the induction of AA release as compared with C₆-C1P, C₁₆-C1P, and C_18:1 C1P. Here, we demonstrate that C1P requires ≥6 carbon acyl-chain to activate cPLA₂α. Thus, published reports on the biological activity of C₂-C1P are not via eicosanoid synthesis. Furthermore, this study demonstrates that the alcohol/dodecane system can be used to efficiently deliver exogenous phospholipids to cells for the examination of specific biological effects.—Wijesinghe, D. S., P. Subramanian, N. F. Lamour, L. B. Gentile, M. H. Granado, A. Bielawska, Z. Szulc, A. Gomez-Munoz, and C. E. Chalfant. Chain length specificity for activation of cPLA₂α by C1P: use of the dodecane delivery system to determine lipid-specific effects.     

Expression of human growth hormone in the milk of transgenic rabbits with transgene mapped to the telomere region of chromosome 7q

The advent of transgenic technology has provided methods for the production of pharmaceuticals by the isolation of these proteins from transgenic animals. The mammary gland has been focused on as a bioreactor, since milk is easily collected from lactating animals and protein production can be expressed at very high levels, including hormones and enzymes. We demonstrate here the expression pattern of recombinant human growth hormone (rhGH) in transgenic rabbits carrying hGH genomic sequences driven by the rat whey acidic protein (WAP) promoter. The transgene was mapped to the q26-27 telomere region of chromosome 7q by fluorescence in situ hybridization (FISH). Nearly 30 % of the F1 generation demonstrated the presence of transgene. The recombinant growth hormone was detected in the milk of the transgenic rabbit females, but not in serum, up to the level of 10???g/ml. Ectopic expression of the transgene in the brain, heart, kidney, liver, and salivary gland was not observed, indicating that a short sequence of rat WAP promoter (969 bp) contained essential sequences directing expression exclusively to the mammary gland. The biological activity of recombinant growth hormone was measured by immunoreactivity and the capability to stimulate growth of the hormone-dependent Nb211 cell line.     

Structural analysis and sheep pituitary receptor binding of GnRH and its complexes with metal ions

Binding of GnRH and its metal complexes to a sheep pituitary receptor have been investigated showing that Cu(II)-GnRH complex is more effectively bound to the receptor than the metal-free ligand, while Ni(II) and Co(II) complexes are less effective than the metal-free GnRH. Earlier studies have explained reasonably well the complex formation with cupric ion, while in this work extensive 1H NMR measurements have been performed for free gonadotropin-releasing hormone (GnRH) and its complexes with Ni(II) in DMSO (dimethyl sulfoxide) solution. This study shows the high order of organization of the metal-free peptide in DMSO solution with two structured 'domains' whose relative orientation is modulated by the mobility of the central glycine. Furthermore, theoretical calculations were performed for the Ni(II)-GnRH complex. The data obtained in this work supports previous studies on the co-ordination of Ni(II) ions with GnRH in aqueous solutions at high pH [J. Inorg. Biochem. 33 (1988) 11] and suggest an experimental procedure to reproduce high pH in DMSO solution. In the Ni(II) complex, the metal ion was found to co-ordinate with four nitrogen atoms inducing a well definite arrangement of aromatic side-chains and a rigid backbone structure.     

Molecular phylogenetic analysis of the Microphalloidea Ward, 1901 (Trematoda: Digenea)

Phylogenetic interrelationships of 32 species belonging to 18 genera and four families of the superfamily Microphalloidea were studied using partial sequences of nuclear lsrDNA analysed by Bayesian inference and maximum parsimony. The resulting trees were well resolved at most nodes and demonstrated that the Microphalloidea, as represented by the present data-set, consists of three main clades corresponding to the families Lecithodendriidae, Microphallidae and Pleurogenidae + Prosthogonimidae. Interrelationships of taxa within each clade are considered; as a result of analysis of molecular and morphological data, Floridatrema Kinsella & Deblock, 1994 is synonymised with Maritrema Nicoll, 1907, Candidotrema Dollfus, 1951 with Pleurogenes Looss, 1896, and Schistogonimus Lühe, 1909 with Prosthogonimus Lühe, 1899. The taxonomic value of some morphological features, used traditionally for the differentiation of genera within the Lecithodendriidae and Prosthogonimidae, is reconsidered. Previous systematic schemes are discussed from the viewpoint of present results, and perspectives of future studies are outlined.