The core domain of retrotransposon integrase in Hordeum: predicted structure and evolution

Propagation of long terminal repeat (LTR)-bearing retrotransposons and retroviruses requires integrase (IN, EC 2.7.7.-), encoded by the retroelements themselves, which mediates the insertion of cDNA copies back into the genome. An active retrotransposon family, BARE-1, comprises approximately 7% of the barley (Hordeum vulgare subsp. vulgare) genome. We have generated models for the secondary and tertiary structure of BARE-1 IN and demonstrate their similarity to structures for human immunodeficiency virus 1 and avian sarcoma virus INs. The IN core domains were compared for 80 clones from 28 Hordeum accessions representative of the diversity of the genus. Based on the structural model, variations in the predicted, aligned translations from these clones would have minimal structural and functional effects on the encoded enzymes. This indicates that Hordeum retrotransposon IN has been under purifying selection to maintain a structure typical of retroviral INs. These represent the first such analyses for plant INs.      

Organization of fusicoccin receptor in higher plant plasma membrane: relationship between affinity and molecular mass

Higher plant plasma membranes carry receptors of different affinity for the phytotoxin fusicoccin. Reception of fusicoccin involves proteins belonging to the highly conserved 14-3-3 family, but the complete structure of the fusicoccin receptor (FCR) is unknown. Using radiation inactivation analysis, we estimated the molecular masses of low-affinity and high-affinity FCR at 63 +/- 7 and 130 +/- 15 kD, respectively. The dose dependences of receptor inactivation indicate that microsomal specimens contain "silent" FCRs of 420 +/- 90 kD in amounts commensurate with that of the active FCRs. Both low- and high-affinity FCRs are inactivated by hydrolytic enzymes from the outer surface of the plasma membrane, and impairment of protoplast integrity causes an irreversible transition of the low-affinity binding site into the high-affinity one. A scheme is proposed for the organization of different types of FCR in the plasma membrane, implying that the membrane affinity for fusicoccin reflects the interaction between proteins in the FCR complex.     

Biotinyl-l-3-(2-naphthyl)-alanine hydrazide derivatives of N-glycans: versatile solid-phase probes for carbohydrate-recognition studies

Biotinyl-oligosaccharides are a relatively new generation of saccharide probes that enable immobilization of desired oligosaccharides on streptavidin matrices for studies of carbohydrate-protein interactions. Here we describe the facile preparation of biotinyl-l-3-(2-naphthyl)- alanine hydrazide (BNAH) derivatives of oligosaccharides, containing a strong UV absorbing and fluorescent group, in which the ring of the reducing-end monosaccharide is nonreduced. We evaluate reactivities of immobilized BNAH- N -glycans with plant lectins that recognize aspects of the oligosaccharide core or outer-arms. We make some comparisons with 2-amino-6-amidobiotinyl-pyridine (BAP) derivatives obtained by reductive amination, and 6-(biotinyl)-aminocaproyl-hydrazide (BACH) derivatives which have a longer spacer-arm. N -Glycan-BNAH and-BAP derivatives have, overall, comparable reactivities with lectins which recognize N -glycan outer-arms or the trimannosyl core, but only BNAH and BACH derivatives are bound by lectins which recognize the non- reduced core. Moreover, with Pisum sativum agglutinin (PSA) which additionally requires the fucosyl- N- glycan-asparaginyl core for high affinity binding, the immobilized BNAH derivative (which is an alanine hydrazide beta-glycoside) can substitute for the natural beta- glycosylasparaginyl core, whereas the BACH derivative (aminocaproyl- hydrazide-beta-glycoside) is less effective. BNAH is a derivatization reagent of choice, therefore, for solid phase carbohydrate-binding experiments with immobilized N -glycans.      

Monophyly of the order Rodentia inferred from mitochondrial DNA sequences of the genes for 12S rRNA, 16S rRNA, and tRNA-valine

A recent analysis of amino acid sequence data (Graur et al.) suggested that the mammalian order Rodentia is polyphyletic, in contrast to most morphological data, which support rodent monophyly. At issue is whether the hystricognath rodents, such as the guinea pig, represent an independent evolutionary lineage within mammals, separate from the sciurognath rodents. To resolve this problem, we sequenced a region (2,645 bp) of the mitochondrial genome of the guinea pig containing the complete 12S ribosomal RNA, 16S ribosomal RNA, and transfer RNA(VAL) genes for comparison with the available sciurognath and other mammalian sequences. Several methods of analysis and statistical tests of the data all show strong support for rodent monophyly (91%-98% bootstrap probability, or BP). Calibration with the mammalian fossil record suggests a Cretaceous date (107 mya) for the divergence of sciurognaths and hystricognaths. An older date (38 mya) for the controversial Mus- Rattus divergence also is supported by these data. Our neighbor-joining analyses of all available sequence data (25 genes) confirm that some individual genes support rodent polyphyly but that tandem analysis of all data does not. We propose that the conflicting results are due to several compounding factors. The unique biochemical properties of some hystricognath metabolic proteins, largely responsible for generating this controversy, may have a single explanation: a cascade effect resulting from inactivation of the zinc-binding abilities of insulin. After excluding six genes possibly affected by insulin inactivation, analyses of all available sequence data (7,117 nucleotide sites, 3,099 amino acid sites) resulted in strong support for rodent monophyly (94% BP for DNA sequences, 90% for protein sequences), which lends support to the insulin-cascade hypothesis.      

Mechanism of estrogen-induced 17-beta-hydroxysteroid dehydrogenase in ovariectomized rat uterus

Estradiol (E2), progesterone or medroxyprogesterone acetate can induce biosynthesis of the 17-beta-hydroxysteroid dehydrogenase (17-beta-HSD) in the mammalian uterus. For further understanding the 17-beta-HSD induction which may be mediated by the conjugation of the E2 to its receptor, premature ovariectomized rats were treated with E2, or with a synthetic steroid, diethylstilbestrol (DES), an agonist for the E2 receptor but not a substrate for 17-beta-HSD. Histological observation and uterus weight were examined as parameters to evaluate uterine response to those hormones at different durations of treatment. The 17-beta-HSD in ovariectomized rat uterus of each group was also examined by histochemical and biochemical assays. The results showed that the 17-beta-HSD activity in the uterus can be induced by E2 or DES, after daily treatment for 1, 14 and 28 days, but much higher in DES treated animals. The uterus weight demonstrated a "negative linear correlation" to the enzyme activity in all E2 treated groups, but not in DES or control rats. Accordingly, it was indicated that the 17-beta-HSD induction was regulated by conjugation of E2 or DES to its receptor. Therefore, we believe that the 17-beta-HSD gene in the rat uterus is another estrogen responsive gene.     

The evolutionary history of Drosophila buzzatii. XXX. Mitochondrial DNA polymorphism in original and colonizing populations

Both original and colonizer populations of Drosophila buzzatii have been analyzed for mtDNA restriction polymorphisms. Most of the mtDNA nucleotide variation in original populations of NW Argentina can be explained by intrapopulation diversity and only a small fraction can be accounted for by between-population diversity. Similar results are obtained using either the estimated number of nucleotide substitutions per site or considering each restriction site as a locus. Colonizer populations of the Iberian Peninsula are monomorphic and show only the most common haplotype from the original populations. Under the infinite island model and assuming that populations are in equilibrium, fixation indices indicate enough gene flow to explain why the populations are not structured. Yet, the possibility exists that populations have not reached an equilibrium after a founder event at the end of the last Pleistocene glaciation. Tajima's test suggests that directional selection and/or a recent bottleneck could explain the present mtDNA differentiation. Considering the significant population structure found for the chromosomal and some allozyme polymorphisms, the among- population uniformity for mtDNA variability argues in favor of the chromosomal and some allozyme polymorphisms being adaptive.      

Mitochondria in gastric epithelial cells are the key targets for NSAIDs-induced injury and NGF cytoprotection

Gastric epithelial cells are important components of mucosal protection and targets of nonsteroidal anti-inflammatory drugs (NSAIDs)-induced injury. Diclofenac (DFN) is one of the most widely used NSAIDs; however, even its short-term use can induce gastric erosions and ulcers. Nerve growth factor (NGF) has been reported to act not only on neuronal cells but also on endothelial cells; however, its action on gastric epithelial cells is unknown. This study was aimed to determine, whether NGF can protect gastric epithelial cells against DFN-induced injury, and to determine the underlying molecular mechanisms with a focus on mitochondria, survivin, and insulin-like growth factor 1 (IGF-1). Cultured normal rat gastric mucosal epithelial cells 1 (RGM1) were treated with phosphate-buffered saline (PBS; control), NGF (100 ng/mL) and/or DFN (0.25-1.00 mM) for 4 hours. We examined: (1) cell injury by confocal microscopy; (2) cell death/survival using Calcein AM live cell tracking dye; (3) mitochondrial structure and membrane potential function using MitoTracker in live cells; and (4) expression of NGF, its receptor - tropomyosin receptor kinase A (TrkA), survivin and IGF-1 by immunostaining. DFN treatment of RGM1 cells for 4 hours caused extensive cell injury, mitochondrial disintegration, reduced cell viability (from 94 ± 3% in controls to 14 ± 4% in 0.5 mM DFN-treated cells; P < 0.001), and expression of survivin and IGF-1. NGF treatment significantly increased survivin and IGF-1 expression by 41% and 75%, respectively versus PBS controls. Pretreatment with NGF before DFN treatment reduced mitochondrial damage and cell death by 73% and 82%, respectively versus treatment with DFN alone (all P < 0.001). This study also showed the presence of high-affinity TrkA receptors in the plasma membrane and mitochondria of RGM1 cells indicating novel actions of NGF.     

Molecular dynamics of synthetic leucine-serine ion channels in a phospholipid membrane

Molecular dynamics calculations were carried out on models of two synthetic leucine-serine ion channels: a tetrameric bundle with sequence (LSLLLSL)(3)NH(2) and a hexameric bundle with sequence (LSSLLSL)(3)NH(2). Each protein bundle is inserted in a palmitoyloleoylphosphatidylcholine bilayer membrane and solvated by simple point charge water molecules inside the pore and at both mouths. Both systems appear to be stable in the absence of an electric field during the 4 ns of molecular dynamics simulation. The water motion in the narrow pore of the four-helix bundle is highly restricted and may provide suitable conditions for proton transfer via a water wire mechanism. In the wider hexameric pore, the water diffuses much more slowly than in bulk but is still mobile. This, along with the dimensions of the pore, supports the observation that this peptide is selective for monovalent cations. Reasonable agreement of predicted conductances with experimentally determined values lends support to the validity of the simulations.