Effects of Syngenta Enogen Feed Corn containing an α-amylase trait on finishing cattle performance and carcass characteristics.

Two experiments evaluated the effects of feeding a new corn hybrid, containing an α-amylase enzyme trait, Syngenta Enogen Feed Corn (SYT-EFC), on feedlot performance and carcass characteristics at two locations. Experiment 1 utilized 300 calffed steers (298.5 ± 16.3 kg of BW) at the University of Nebraska-Lincoln Eastern Nebraska Research and Extension Center Mead, NE. Treatments were designed as a 2 × 2 + 1-factorial arrangement with factors consisting of 1) corn type (SYT-EFC or conventional [CON]) and 2) byproduct type (with or without Sweet Bran [SB]), or a BLEND of STY-EFC and CON without SB. In Exp. 2, 240 crossbred, calf-fed steers (287.6 ± 15.4 kg of BW) were utilized at the University of Nebraska-Lincoln Panhandle Research and Extension Center near Scottsbluff, NE. Steers were fed SYT-EFC, CON, BLEND, or CON with a commercial α-amylase enzyme supplement (CON-E). In Exp. 1, there was an interaction for ADG (P = 0.05) and G:F (P = 0.02). Steers fed SYT-EFC with SB had greater ADG and G:F than CON; however, in diets without SB, SYT-EFC and CON were not different resulting in a 10.1% change in G:F when steers were fed SYT-EFC in SB compared with CON and only 1.6% change between SYT-EFC and CON without SB. Energy values, based on performance data, resulted in a 6.5% and 8.3% change in NEm and NEg, respectively, for steers fed SYT-EFC and CON with SB and 1.6% change for both NEm and NEg for steers fed SYT-EFC and CON without SB. For the main effect of corn trait, steers fed SYT-EFC had greater marbling scores, fat depth, and calculated yield grade compared with CON (P ≤ 0.03). In diets without SB, there was no difference between SYT-EFC, CON, or BLEND for DMI, final BW, ADG, G:F, NEm, or NEg (P ≥ 0.35). In Exp. 2, cattle fed SYT-EFC, BLEND, or CON-E had greater final BW, ADG, and G:F than cattle fed CON (P ≤ 0.03). On average, NEm and NEg were 4.9% and 7.0% greater, respectively, for steers fed amylase enzyme treatments compared with CON (P ≤ 0.01). Hot carcass weights were greater in steers fed α-amylase treatments compared with CON (P < 0.01). Feeding Syngenta Enogen Feed Corn, which contains an α-amylase enzyme trait, at both locations improved feed efficiency in finishing cattle diets containing WDGS or SB.

Discrimination in resolving systems. VI. Comparison of the diastereomers of deoxyephedrinium and ephedrinium 4'-fluoromandelates.

(-)-(R)-Deoxyephedrine forms poorly discriminating diastereomeric salts with 4'-fluoromandelic acid from 95% ethanol. Both less-soluble (L) (S)-4'-fluoromandelate and more-soluble (M) (R)-4'-fluoromandelate phases are monoclinic and unsolvated. Their solubility ratio (M/L) in 95% ethanol is only 1.2, which correlates with the similarity and small differences in their respective heats of fusion and fusion temperatures. The (R)-deoxyephedrinium and the related (1R;2S)-ephedrinium 4'-fluoromandelate systems show L-salts with higher ion-pair volumes and lower densities than their M-salts. (R)-Deoxyephedrinium salts have higher volumes than the comparable (1R;2S)-ephedrinium salts even though the resolving base (R)-deoxyephedrine lacks the benzylic hydroxy. In the solids, bilayered structures segregate polar and nonpolar molecular regions. The principle interionic interactions are hydrogen bonds between protonated secondary ammonium ions and carboxylates forming infinite chains with a six-atom repeating unit H-N(+)-H...O-C(-)-O [C(2)(2)(6)]. These are buttressed by mandelate hydroxy to carboxylate hydrogen bonds. Differing interactions between phenyl and 4'-fluorophenyl rings in the nonpolar layers of the salts correlate with the density and stability inversion.

Helix P4 is a divalent metal ion binding site in the conserved core of the ribonuclease P ribozyme.

The ribonuclease P ribozyme (RNase P RNA), like other large ribozymes, requires magnesium ions for folding and catalytic function; however, specific sites of metal ion coordination in RNase P RNA are not well defined. To identify and characterize individual nucleotide functional groups in the RNase P ribozyme that participate in catalytic function, we employed self-cleaving ribozyme-substrate conjugates that facilitate measurement of the effects of individual functional group modifications. The self-cleavage rates and pH dependence of two different ribozyme-substrate conjugates were determined and found to be similar to the single turnover kinetics of the native ribozyme. Using site-specific phosphorothioate substitutions, we provide evidence for metal ion coordination at the pro-Rp phosphate oxygen of A67, in the highly conserved helix P4, that was previously suggested by modification-interference experiments. In addition, we detect a new metal ion coordination site at the pro-Sp phosphate oxygen of A67. These findings, in combination with the proximity of A67 to the pre-tRNA cleavage site, support the conclusion that an important role of helix P4 in the RNase P ribozyme is to position divalent metal ions that are required for catalysis.

A block to human immunodeficiency virus type 1 assembly in murine cells.

Human immunodeficiency virus type 1 (HIV-1) does not replicate in murine cells. We investigated the basis of this block by infecting a murine NIH 3T3 reporter cell line that stably expressed human CD4, CCR5, and cyclin T1 and contained a transactivatable HIV-1 long terminal repeat (LTR)-green fluorescent protein (GFP) cassette. Although the virus entered efficiently, formed provirus, and was expressed at a level close to that in a highly permissive human cell line, the murine cells did not support M-tropic HIV-1 replication. To determine why the virus failed to replicate, the efficiency of each postentry step in the virus replication cycle was analyzed using vesicular stomatitis virus G pseudotypes. The murine cells supported reverse transcription and integration at levels comparable to those in the human osteosarcoma-derived cell line GHOST.R5, and human cyclin T1 restored provirus expression, consistent with earlier findings of others. The infected murine cells contained nearly as much virion protein as did the human cells but released less than 1/500 the amount of p24(gag) into the culture medium. A small amount of p24(gag) was released and was in the form of fully infectious virus. Electron microscopy suggested that aberrantly assembled virion protein had accumulated in cytoplasmic vesicular structures. Virions assembling at the cell membrane were observed but were rare. The entry of M-tropic JR.FL-pseudotyped reporter virus was moderately reduced in the murine cells, suggesting a minor reduction in coreceptor function. A small reduction in the abundance of full-length viral mRNA transcripts was also noted; however, the major block was at virion assembly. This could have been due to a failure of Gag to target to the cell membrane. This block must be overcome before a murine model for HIV-1 replication can be developed.

RNA export: insights from viral models.

The retroviruses export intron-containing RNA. The complex retroviruses encode a Rev protein that uses a leucine-rich NES to interact with CRM1 and the U snRNA-export pathway. Other viruses encode proteins with a Rev-like NES. The type-D retroviruses contain a CTE that binds the cellular protein TAP to export intron-containing RNA through the mRNA pathway. Intronless viral transcripts contain post-transcriptionally acting RNA elements that may compensate for the lack of an intron. The functions of elements in intronless RNA are not fully understood but may be in export and/or 3'-end processing.

The track of the pre-tRNA 5' leader in the ribonuclease P ribozyme-substrate complex.

The ribonuclease P (RNase P) ribozyme is an endonuclease that binds precursor tRNAs and catalyzes the removal of 5' leader nucleotides. Biochemical and photo-cross-linking studies have identified sites of contact between the mature tRNA domain of pre-tRNA and the ribozyme; however, relatively little is known about the location of the 5' leader in the ribozyme-substrate complex. To investigate the local three-dimensional environment of the 5' leader, we employed the short-range photo-cross-linking agent 4-thiouridine (s(4)U). The s(4)U photoagent was incorporated into a series of pre-tRNA substrates containing unique uridine residues in the 5' leader sequence at positions -1, -3, -5, -7, or -10. The modified substrates formed high-affinity complexes with the ribozyme and produced discrete intermolecular cross-links to RNase P RNA from Bacillus subtilis. Locations of the cross-linked nucleotides in the ribozyme and pre-tRNA were determined by reverse transcriptase primer extension. Photoagents incorporated into the 5' leader detected discrete elements of ribozyme structure in a progression from J18/2 to L15 to P3. Importantly, all of the cross-linked species retained the ability to cleave the covalently attached pre-tRNA, indicating that the cross-links reflect the native structure of the ribozyme-substrate complex. Together with available structural and biochemical data, the cross-linking results suggest a model for the position of the 5' leader within the ground-state ribozyme-substrate complex.

Enhanced expression of transgenes from adeno-associated virus vectors with the woodchuck hepatitis virus posttranscriptional regulatory element: implications for gene therapy.

The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) evolved to stimulate the expression of intronless viral messages. To determine whether this ability to enhance expression could be useful in nonviral and heterologous viral gene delivery systems, we analyzed the ability of the WPRE to elevate the expression of a cDNA encoding the green fluorescent protein (GFP) in these contexts. We find that the WPRE can stimulate the expression of GFP when the gene is delivered by transfection or transduction with recombinant adeno-associated virus (AAV). Enhancement occurred both during transient expression and when the gene is stably incorporated into the genome of target cells. This enhancement required that the WPRE be located in cis within the GFP message, and was observed in both transformed cell lines and primary human fibroblasts. These results demonstrate that the WPRE will be an effective tool for increasing the long-term expression of transgenes in gene therapy.

Use of circular permutation and end modification to position photoaffinity probes for analysis of RNA structure.

Photocrosslinking allows first-order structural analysis with relatively small amounts of biological material and can be applied in complex in vitro systems. In this article we describe methods for positioning both arylazide and thionucleotide photoagents within an RNA of interest by end modification of circularly permuted RNAs. Application of this technique provided a library of constraints that, together with biochemical and phylogenetic comparative data, were used to develop a structure model of the bacterial ribonuclease P ribozyme-substrate complex. Circularly permuted genes for in vitro transcription are generated by PCR from tandem genes. Circularly permuted RNA transcripts can be modified with high efficiency at both the 5' and 3' termini with arylazide crosslinking reagents, or transcription can be primed with photoactive nucleotide analog monophosphates such as 6-thioguanosine. These crosslinking agents can be used over a wide range of experimental conditions but remain inert until they are activated by UV light. Crosslinked sites are subsequently mapped by reverse transcriptase primer extension of gel-purified crosslinked species. In addition to providing basic protocols for these methods, we discuss approaches for establishing the relevance of crosslinking data to native RNA structure.

Identification of adenosine functional groups involved in substrate binding by the ribonuclease P ribozyme.

The RNA component of bacterial ribonuclease P (RNase P) binds to substrate pre-tRNAs with high affinity and catalyzes site-specific phosphodiester bond hydrolysis to generate the mature tRNA 5' end. Herein we describe the use of biotinylated pre-tRNA substrates to isolate RNase P ribozyme-substrate complexes for nucleotide analogue interference mapping of ribozyme base functional groups involved in substrate recognition. By using a series of adenosine base analogues tagged with phosphorothioate substitutions, we identify specific chemical groups involved in substrate binding. Only 10 adenosines in the Escherichia coli ribozyme show significant sensitivity to interference: A65, A66, A136, A232-234, A248, A249, A334, and A347. Most of these adenosine positions are universally conserved among all bacterial RNase P RNAs; however, not all conserved adenosines are sensitive to analogue substitution. Importantly, all but one of the sensitive nucleotides are located at positions of intermolecular cross-linking between the ribozyme and the substrate. One site of interference that did not correlate with available structural data involved A136 in J11/12. To confirm the generality of the results, we repeated the interference analysis of J11/12 in the Bacillus subtilis RNase P ribozyme, which differs significantly in overall secondary structure. Notably, the B. subtilis ribozyme shows an identical interference pattern at the position (A191) that is homologous to A136. Furthermore, mutation of A136 in the E. coli ribozyme gives rise to a measurable increase in the equilibrium binding constant for the ribozyme-substrate interaction, while mutation of a nearby conserved nucleotide (A132) that is not sensitive to analogue incorporation does not. These results strongly support direct participation of nucleotides in the P4, P11, J5/15, and J18/2 regions of ribozyme structure in pre-tRNA binding and implicate an additional region, J11/12, as involved in substrate recognition. In aggregate, the interference results provide a detailed chemical picture of how the conserved nucleotides adjacent to the pre-tRNA substrate contribute to substrate binding and provide a framework for subsequent identification of the specific roles of these chemical groups in substrate recognition.

Identification of individual nucleotides in the bacterial ribonuclease P ribozyme adjacent to the pre-tRNA cleavage site by short-range photo-cross-linking.

The bacterial RNase P ribozyme is a site-specific endonuclease that catalyzes the removal of pre-tRNA leader sequences to form the 5' end of mature tRNA. While several specific interactions between enzyme and substrate that direct this process have been determined, nucleotides on the ribozyme that interact directly with functional groups at the cleavage site are not well-defined. To identify individual nucleotides in the ribozyme that are in close proximity to the pre-tRNA cleavage site, we introduced the short-range photoaffinity cross-linking reagent 6-thioguanosine (s6G) at position +1 of tRNA and position -1 in a tRNA bearing a one-nucleotide leader sequence [tRNA(G-1)] and examined cross-linking in representatives of the two structural classes of bacterial RNase P RNA (from Escherichia coli and Bacillus subtilis). These photoagent-modified tRNAs bind with similar high affinity to both ribozymes, and the substrate bearing a single s6G upstream of the cleavage (-1) site is cleaved accurately. Interestingly, s6G at position +1 of tRNA cross-links with high efficiency to homologous positions in J5/15 in both E. coli and B. subtilis RNase P RNAs, while s6G at position -1 of tRNA(G-1) cross-links to homologous nucleotides in J18/2. Both cross-links are detected over a range of ribozyme and substrate concentrations, and importantly, ribozymes cross-linked to position -1 of tRNA(G-1) accurately cleave the covalently attached substrate. These data indicate that the conserved guanosine at the 5' end of tRNA is adjacent to A248 (E. coli) of J5/15, while the base upstream of the substrate phosphate is adjacent to G332 (E. coli) of J18/2 and, along with available biochemical data, suggest that these nucleotides play a direct role in binding the substrate at the cleavage site.

Leptomycin B inhibits equine infectious anemia virus Rev and feline immunodeficiency virus rev function but not the function of the hepatitis B virus posttranscriptional regulatory element.

Human immunodeficiency virus type 1 Rev export depends upon the presence of the nuclear export signal (NES), a leucine-rich stretch of hydrophobic amino acids. Recently, the nuclear NES-binding receptor has been identified as CRM1 or exportin 1. Rev export has been shown to be CRM1 dependent. The function of the atypical NES-containing Rev-like proteins of equine infectious anemia virus (EIAV) and feline immunodeficiency virus (FIV) is inhibited by leptomycin B, a drug that specifically blocks NES-CRM1 interactions. These data suggest that the function of atypical NES-containing proteins is CRM1 dependent. In contrast to the inhibition of EIAV Rev and FIV Rev, the cytoplasmic accumulation of hepatitis B virus (HBV) posttranscriptional regulatory element (PRE)-containing and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-containing RNAs is not inhibited by leptomycin B treatment. We conclude that the HBV PRE, like the MPMV CTE, functions independently of an NES receptor-exportin 1 interaction.

Differential requirements for alternative splicing and nuclear export functions of equine infectious anemia virus Rev protein.

The Rev protein of equine infectious anemia virus (ERev) exports unspliced and partially spliced viral RNAs from the nucleus. Like several cellular proteins, ERev regulates its own mRNA by mediating an alternative splicing event. To determine the requirements for these functions, we have identified ERev mutants that affect RNA export or both export and alternative splicing. Mutants were further characterized for subcellular localization, nuclear-cytoplasmic shuttling, and multimerization. None of the nuclear export signal (NES) mutants are defective for alternative splicing. Furthermore, the NES of ERev is similar in composition but distinct in spacing from other leucine-rich NESs. Basic residues at the C terminus of ERev are involved in nuclear localization, and disruption of the C-terminal residues affects both functions of ERev. ERev forms multimers, and no mutation disrupts this activity. In two mutants with substitutions of charged residues in the middle of ERev, RNA export is affected. One of these mutants is also defective for ERev-mediated alternative splicing but is identical to wild-type ERev in its localization, shuttling, and multimerization. Together, these results demonstrate that the two functions of ERev both require nuclear import and at least one other common activity, but RNA export can be separated from alternative splicing based on its requirement for a functional NES.

Comparative photocross-linking analysis of the tertiary structures of Escherichia coli and Bacillus subtilis RNase P RNAs.

Bacterial ribonuclease P contains a catalytic RNA subunit that cleaves precursor sequences from the 5' ends of pre-tRNAs. The RNase P RNAs from Bacillus subtilis and Escherichia coli each contain several unique secondary structural elements not present in the other. To understand better how these phylogenetically variable elements affect the global architecture of the ribozyme, photoaffinity cross-linking studies were carried out. Photolysis of photoagents attached at homologous sites in the two RNAs results in nearly identical cross-linking patterns, consistent with the homology of the RNAs and indicating that these RNAs contain a common, core tertiary structure. Distance constraints were used to derive tertiary structure models using a molecular mechanics-based modeling protocol. The resulting superimposition of large sets of equivalent models provides a low resolution (5-10 A) structure for each RNA. Comparison of these structure models shows that the conserved core helices occupy similar positions in space. Variably present helical elements that may play a role in global structural stability are found at the periphery of the core structure. The P5.1 and P15.1 helical elements, unique to the B.subtilis RNase P RNA, and the P6/16/17 helices, unique to the E.coli RNA, occupy similar positions in the structure models and, therefore, may have analogous structural function.

Biological characterization of Rev variation in equine infectious anemia virus.

Sequence analysis identified significant variation in the second exon of equine infectious anemia virus (EIAV) rev. Functional analysis indicated that limited amino acid variation in Rev significantly altered the export activity of the protein but did not affect Rev-dependent alternative splicing. EIAV Rev can mediate export through two independent cis-acting Rev-responsive elements (RREs), and differences among Rev variants were more pronounced when both RREs were present. Variation in Rev may be an important mechanism for regulation of virus replication in vivo and may contribute to changes in clinical disease.

Analysis of the tertiary structure of the ribonuclease P ribozyme-substrate complex by site-specific photoaffinity crosslinking.

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well-established, and a low-resolution model of the three-dimensional structure of the ribozyme-substrate complex has been proposed based on site-specific crosslinking and phylogenetic comparative data [Harris ME et al., 1994 EMBO J 13:3953-3963]. However, several substructures of that model were poorly constrained by the available data. In the present analysis, additional constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments. Circularly permuted RNase P RNAs were used to position an azidophenacyl photoactive crosslinking agent specifically at strategic sites within the ribozyme-substrate complex. Crosslink sites were mapped by primer extension and confirmed by analysis of the mobility of the crosslinked RNA lariats on denaturing acrylamide gels relative to circular and linear RNA standards. Crosslinked species generally retained significant catalytic activity, indicating that the results reflect the native ribozyme structure. The crosslinking results support the general configuration of the structure model and predicate new positions and orientations for helices that were previously poorly constrained by the data set. The expanded library of crosslinking constraints was used, together with secondary and tertiary structure identified by phylogenetic sequence comparisons, to refine significantly the model of RNase P RNA with bound substrate pre-tRNA. The crosslinking results and data from chemical-modification and mutational studies are discussed in the context of the current structural perspective on this ribozyme.

Amyloid beta peptide (25-35) inhibits Na+-dependent glutamate uptake in rat hippocampal astrocyte cultures.

Large numbers of neuritic plaques surrounded by reactive astrocytes are characteristic of Alzheimer's disease (AD). There is a large body of research supporting a causal role for the amyloid beta peptide (Abeta), a main constituent of these plaques, in the neuropathology of AD. Several hypotheses have been proposed to explain the toxicity of Abeta including free radical injury and excitotoxicity. It has been reported that treatment of neuronal/astrocytic cultures with Abeta increases the vulnerability of neurons to glutamate-induced cell death. One mechanism that may explain this finding is inhibition of the astrocyte glutamate transporter by Abeta. The aim of the current study was to determine if Abetas inhibit astrocyte glutamate uptake and if this inhibition involves free radical damage to the transporter/astrocytes. We have previously reported that Abeta can generate free radicals, and this radical production was correlated with the oxidation of neurons in culture and inhibition of astrocyte glutamate uptake. In the present study, Abeta (25-35) significantly inhibited L-glutamate uptake in rat hippocampal astrocyte cultures and this inhibition was prevented by the antioxidant Trolox. Decreases in astrocyte function, in particular L-glutamate uptake, may contribute to neuronal degeneration such as that seen in AD. These results lead to a revised excitotoxicity/free radical hypothesis of Abeta toxicity involving astrocytes.

Enhancement of beta-amyloid peptide A beta(1-40)-mediated neurotoxicity by glutamine synthetase.

The beta-amyloid peptide (A beta), a main constituent in both senile and diffuse plaques in Alzheimer's disease brains, was previously shown to be neurotoxic and to be able to interact with several macromolecular components of brain tissue. Previous investigations carried out in our laboratory demonstrated free radical species formation in aqueous solutions of A beta(1-40) and its C-end fragment, A beta(25-35). Toxic forms of A beta rapidly inactivate the oxidation-sensitive cytosolic enzyme glutamine synthetase (GS). In this regard, we suggested and subsequently demonstrated that A beta radicals can cause an oxidative damage of cell proteins and lipids resulting in disruption of membrane functions, enzyme inactivation, and cell death. Because GS can be a substrate for A beta-derived oxidizing species, the present study was conducted to determine if GS could protect against A beta neurotoxicity. In contrast to this initial hypothesis, we here report that GS significantly enhances the neurotoxic effects of A beta(1-40). The A beta-mediated inactivation of GS was found to be accompanied by the loss of immunoreactive GS and the significant increase of A beta(1-40) neurotoxicity.

beta-Amyloid peptide-derived, oxygen-dependent free radicals inhibit glutamate uptake in cultured astrocytes: implications for Alzheimer's disease.

beta-Amyloid (A beta), the central constituent of senile plaques in Alzheimer's disease (AD) brains, was shown by us recently to generate free radicals in an oxygen dependent mechanism. A beta-derived free radicals were detected directly using electron paramagnetic resonance (EPR) spin trapping techniques employing the spin trap phenyl-alpha-tert-butylnitrone (PBN). We have extended these studies to investigate the nature of the oxyradicals derived from A beta peptides, and we show that these free radicals are able to inhibit glutamate uptake in cultured astrocytes. An implication of inhibited astrocyte glutamate uptake in brain is increased extracellular levels of glutamate, which is excitotoxic to neurons. These results support the hypothesis that A beta neurotoxicity in AD may be due in part to A beta-derived, oxygen-dependent free radical inhibition of glutamate uptake.

Editing domains of Trypanosoma brucei mitochondrial RNAs identified by secondary structure.

The posttranscriptional insertion and deletion of U residues in trypanosome mitochondrial transcripts called RNA editing initiates at the 3' end of precisely defined editing domains that can be identified independently of the cognate guide RNA. The regions where editing initiates in Trypanosoma brucei cytochrome b and cytochrome oxidase subunit II preedited mRNAs are specifically cleaved by a trypanosome mitochondrial endonuclease that acts like mung bean nuclease and therefore is single strand specific. The regions where editing initiates in virtually all examined preedited mRNAs are predicted to form loop structures, suggesting that editing domains could generally be recognized as prominent single-stranded loops. In contrast to preedited mRNA, edited mRNA can be either resistant or sensitive to cleavage by trypanosome mitochondrial endonuclease, depending on the reaction conditions. This selectivity appears dependent on the availability of extract RNAs, and in model reactions, edited mRNA becomes resistant to cleavage upon base pairing with its guide RNA. Natural partially edited mRNAs are also specifically cleaved with a sensitivity like preedited and unlike edited mRNAs, consistent with their being intermediates in editing. These results suggest that in vivo, the structure of editing domains could initially be recognized by the mitochondrial endonuclease, which could target its associated RNA ligase and terminal U transferase to begin cycles of enzymatic editing modifications.