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.

Modification of Trypanosoma brucei mitochondrial rRNA by posttranscriptional 3' polyuridine tail formation.

Trypanosoma brucei mitochondrial transcripts can be posttranscriptionally processed by uridine addition or deletion. With editing of mRNAs, uridine addition and deletion create precisely altered reading frames. The addition of nonencoded uridines to mitochondrial guide RNAs results in a less precise modification. Although uridines are specifically added to the 3' termini, their number varies, which results in heterogeneous oligo(U) tails on guide RNAs. In this paper, we show that the mitochondrial 9S and 12S rRNAs are also modified by uridine addition. These modifications appear to have aspects in common with both RNA editing and oligo(U) tail formation. Metabolic labeling studies with intact mitochondria and [alpha-32P]UTP, in the absence of transcription, demonstrated the posttranscriptional timing of the event. T1 RNase comparison analyses of cytidine 3',5'-[5'-32P]biphosphate 3'-end-labeled and [alpha-32P]UTP metabolically labeled rRNAs, along with direct RNA sequencing of the 3' termini, identified the site of uridine addition and revealed the creation of an oligo(U) tail for both rRNAs. 12S and 9S rRNAs hybrid selected from total cell RNA exhibited the same modification, demonstrating the presence of this processing in vivo. Moreover, only 3'-poly(U)-tailed 9S and 12S rRNAs were detected in total cellular and mitochondrial RNAs, which suggests that they are the most abundant and probable mature forms. The 12S and 9S rRNA oligo(U) tails differed significantly from each other, with the 12S having a heterogeneous tail of 2 to 17 uridines and the 9S having a tail of precisely 11 uridines. The mechanism of formation and the function of the rRNA poly(U) tails remain to be determined.

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.

Regulation of histone mRNA in the unperturbed cell cycle: evidence suggesting control at two posttranscriptional steps.

The levels of histone mRNA increase 35-fold as selectively detached mitotic CHO cells progress from mitosis through G1 and into S phase. Using an exogenous gene with a histone 3' end which is not sensitive to transcriptional or half-life regulation, we show that 3' processing is regulated as cells progress from G1 to S phase. The half-life of histone mRNA is similar in G1- and S-phase cells, as measured after inhibition of transcription by actinomycin D (dactinomycin) or indirectly after stabilization by the protein synthesis inhibitor cycloheximide. Taken together, these results suggest that the change in histone mRNA levels between G1- and S-phase cells must be due to an increase in the rate of biosynthesis, a combination of changes in transcription rate and processing efficiency. In G2 phase, there is a rapid 35-fold decrease in the histone mRNA concentration which our results suggest is due primarily to an altered stability of histone mRNA. These results are consistent with a model for cell cycle regulation of histone mRNA levels in which the effects on both RNA 3' processing and transcription, rather than alterations in mRNA stability, are the major mechanisms by which low histone mRNA levels are maintained during G1.

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.

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.

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.

Maintenance of rate testis fluid testosterone and dihydrotestosterone levels by pregnenolone and other C21 steroids in hypophysectomized rats.

This study was undertaken to determine whether maintenance of spermatogenesis in hypophysectomized rats by pregnenolone and other C21 steroids may be due to in vivo conversion of these compounds to androgens. Hypophysectomized rats were treated sc with 2 mg of pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone or testosterone propionate in 0.2 ml sesame oil daily for 14 days beginning 2 days after hypophysectomy. Rete testis fluid (RTF), peripheral blood, and testicular venous blood were collected on the day of the last injection. Testosterone (T) and dihydrotestosterone (DHT) were measured by radioimmunoassay after chromatographic separation. Results demonstrate that T and DHT could be found in the RTF of C21 steroid-treated hypophysectomized rats at levels similar to those seen in the intact rat. Results imply that the maintenance of spermatogenesis by C21 steroids is probably due to the conversion of these compounds to T in the testis. Relatively little T was released from the testis into the peripheral circulation of these rats since T levels in testicular venous plasma were low and peripheral plasma T levels were not distinguishable from those seen in untreated hypophysectomized rats. Histological examination of the testes of C21 steroid-treated hypophysectomized rats showed nearly quantitative maintence of spermatogenesis and atrophy of Leydig cells. These findings suggest that most of the conversion of C21 steroids to androgens occurred in the seminiferous tubules.

Trypanosoma brucei mitochondrial ribosomal RNA synthesis, processing and developmentally regulated expression.

The steady-state levels of the mitochondrial ribosomal RNAs of Trypanosoma brucei are repressed in the early bloodstream developmental stage of the parasite and accumulate approximately 30-fold during differentiation to the stage found in the midgut of the insect vector. In order to determine the mechanism regulating this developmental process, we have examined the transcription and processing of the 9S and 12S mitochondrial rRNAs of T. brucei. A short-lived RNA was detected in pulse labeling experiments which contains the mature 12S and 9S rRNAs and at least 1200 nucleotides of RNA transcribed from upstream of the 12S rRNA gene. This putative processing precursor RNA was identified in both intact cells and in run-on experiments using isolated mitochondria. The transcripts containing the upstream sequences are unstable and reach isotopic equilibrium within 15 min. Mature rRNAs in the insect developmental stage are stable and show no detectable turnover during a 36-h chase. Comparison of rRNA synthesis in bloodstream and insect life-stages indicates that mitochondrial rRNA levels are controlled not at the transcriptional level, but rather by a mechanism which likely modulates the stability of the mature rRNAs. These results suggest that a short-lived rRNA precursor is synthesized and processed at comparable rates in both bloodstream and insect stages of the parasite. Thus, it appears that differential stability of the mature 9S and 12S rRNAs plays a major role in modulating mitochondrial gene expression during the developmental cycle of T. brucei.

Two-stage tuberculin testing with control antigens in patients residing in two chronic disease hospitals.

We studied the prevalence of tuberculin reactivity and anergy in 360 elderly patients residing in two municipal chronic disease hospitals. Eighty-five (26%) of the 323 patients tested had a positive reaction to a stage 1 tuberculin test and 12 (6%) of the 207 stage 1 tuberculin-negative patients exhibited a booster response to a stage 2 tuberculin test. Thirty percent of the same 207 patients had no response to an anergy panel of skin test antigens that included candida, mumps, and trichophyton. Nonresponders to tuberculin and the anergy panel had significantly higher one-year mortality rates compared to responders (44 v 20%, P = 0.001). Tuberculin-positivity among the 770 employees working in these facilities was 43%; 12 (4%) had a booster response. A survey of 29 randomly selected long-term care facilities in the Boston area indicated that all had a policy for pre-employment screening of employees, but less than 50% had a policy for patients and only one institution used two-stage testing. Routine tuberculin testing is recommended for long-term care facilities and the two-stage method is preferable in institutions with adequate resources.

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.

Effects of testosterone and dihydrotestosterone on spermatogenesis, rete testis fluid, and peripheral androgen levels in hypophysectomized rats.

To compare the effects of testosterone (T) and dihydrotestosterone (DHT) on the maintenance and the restoration of spermatogenesis, hypophysectomized (APX) rats were treated daily for 35 days with 0.5 mg of T propionate (TP) or DHT propionate (DHTP) beginning 5 or 33 days after hypophysectomy. In the maintenance experiment, the weights of the testes and the number of early spermatids were significantly lower in DHTP-than in TP-treated animals, while late spermatids were present only in rats treated with TP. In the restoration experiment, TP increased testicular weight and the number of germinal cells, whereas DHTP had very little effect on the testis. In an attempt to explain these findings, we measured androgen levels in the rete testis fluid (RTF) and peripheral plasma of APX rats treated with TP or DHTP. The concentration of T in the RTF of TP-treated rats was nearly 3-fold higher than the level of DHT in the RTF of animals given DHTP. Plasma T levels measured 1/2, 2, 4, and 24 hours after the last of three daily injections of TP were considerably higher than were the corresponding plasma DHT levels in animals given DHTP. In animals treated with free steroids, peripheral androgen levels between 1/2 and 4 hours after the last injection were much higher in rats given T than in those given DHT, but thereafter this difference disappeared. We conclude that the difference in the ability of subcutaneously injected TP and DHTP to maintain and to restore spermatogenesis in APX rats was due to a difference in androgen levels in the testes of these animals.

Molecular modeling to predict the structural and biological effects of mutations in a highly conserved histone mRNA loop sequence.

The 3'-end of histone mRNAs contains a highly conserved sequence motif which is believed to form a 6 base pair stem and a 4 base loop. These sequences are involved in both the efficiency of 3'-end formation and stability of the mature histone mRNA. We have modeled four stem basepairs and the loop portion of this structure using the wildtype sequences and several mutant sequences. A structure for the wildtype stem-loop is proposed that is based on energy minimization using a representative wildtype sequence and comparison with structures obtained using naturally occurring mutations which do not alter loop function. A wildtype structure is proposed in which the top basepair of the stem is broken, forming a six base loop. Mutant sequences with altered bases in the loop and in the stem were also modeled. The effect of these mutations on the proposed wildtype structure is discussed and possible biological consequences considered.

Techniques of peripheral nerve repair.

At present, the principles of microsurgical reconstruction of the peripheral nerve incorporate a clear understanding of the pathophysiology of the peripheral nerve, accurate preoperative assessment of the lesion, aggressive early treatment to avoid irreversible atrophy of the end organ, use of nontraumatic microtechniques for optimal alignment of individual fascicular bundles, introduction of a minimum amount of foreign material at the suture line, resection of the scar-producing epineurium, total avoidance of tension at the suture line, and placement of the nerve repair in a well-vascularized soft tissue bed. If tension is eliminated, a minimal amount of suture material is required to repair the nerve ends, because the bundles are maintained in anatomical alignment by a fibrin clot. We have reviewed the various nerve repair methods, stressing that with strict attention to microsurgical technique, the surgeon can hope to maximize reinnervation. Although the importance of all aspects of careful surgical technique cannot be overemphasized, we believe that it is unlikely that improved clinical results will come from further refinements in microsurgical techniques. We are not limited by a working knowledge and understanding of the details of the neurobiology and the neurochemistry of nerve regeneration.

Long-term results in the treatment of hydrocephalus.

The majority of infants presenting with progressive hydrocephalus are now treated surgically with ventricular shunting. It is difficult to predict the eventual developmental outcome of most treated patients. This article reviews the results of surgical therapy for infantile hydrocephalus with particular attention to neurologic and intellectual outcome.

Dream content and its relation to self-reported interpersonal behavior.

THE NATURE of the relationship between those psychological processes which influence waking behavior and cognition, and those which influence the content of nocturnal dreams, is a question both interesting and unresolved. Is a person's approach to life similar in both the dream and the waking state? If someone was experiencing conflict in his life, might we expect to find conflictual situations in his dreams? And if, on the contrary, a person's waking expectations and experiences were harmonious, might we expect him to manifest conflict-free dreams? These are the questions to which we addressed ourselves in the present study.