The ambush hypothesis: hidden stop codons prevent off-frame gene reading

Coding sequences lack stop codons, but many stops appear off-frame. Off-frame stops (stops in -1 and +1 shifted reading frames, termed hidden stops) terminate frame-shifted translation, potentially decreasing energy, and resource waste on nonfunctional proteins. Benefits may include reduced waste elimination costs and avoidance of potentially cytotoxic frame-shifted products. Our "ambush" hypothesis suggests that hidden stops are sometimes selected for. Codons of many amino acids can contribute to hidden stops, depending on the synonymous position state and adjacent codons. In vertebrate mitochondria, 31.75% of all amino acid combinations can form hidden stops. Codons with more potential to form hidden stops have greater usage frequency and bias in their favor among synonymous codons. Among primates, predicted mitochondrial rRNA secondary structure stability correlates negatively with the number of hidden stops in the mitochondrial genome. The taxonomic distribution of genetic codes suggests that +1 frameshifts might be more frequent than -1 frameshifts. This is confirmed by analyses of primate mitochondrial genomes: species with unstable rRNAs have more +1 stops, but the correlation is weak for -1 stops. High hidden stop density seems to be an adaptation in species with slippage prone ribosomes (unstable rRNAs). Hidden stops may thus compensate for reduced efficiency of some parts of the biosynthetic machinery. Some experimental data confirm our hypothesis: gene expression increases with the experimentally manipulated number of stops in the promoter region of a gene, suggesting biotechnological applications.     

High glucose and endothelial cell growth: novel effects independent of autocrine TGF-beta 1 and hyperosmolarity

Human endothelial cells wereexposed to 5 mM glucose (control), 25 mM (high) glucose, or osmoticcontrol for 72 h. TGF-1 production, cell growth, death, andcell cycle progression, and the effects of TGF-1 and TGF-neutralization on these parameters were studied. High glucose andhyperosmolarity increased endothelial TGF-1 secretion(P < 0.0001) and bioactivity (P < 0.0001). However, high glucose had a greater effect on reducingendothelial cell number (P < 0.001) and increasingcellular protein content (P < 0.001) than the osmoticcontrol. TGF- antibody only reversed the antiproliferative andhypertrophic effects of high glucose. High glucose altered cell cycleprogression and cyclin-dependent kinase inhibitor expressionindependently of hyperosmolarity. High glucose increased endothelialcell apoptosis (P < 0.01), whereashyperosmolarity induced endothelial cell necrosis (P < 0.001). TGF- antibody did not reverse the apoptotic effectsobserved with high glucose. Exogenous TGF-1 mimicked the increased Sphase delay but not endoreduplication observed with high glucose. High glucose altered endothelial cell growth, apoptosis, and cellcycle progression. These growth effects occurred principally via aTGF-1 autocrine pathway. In contrast, apoptosis andendoreduplication occurred independently of this cytokine and hyperosmolarity.

     

Functional NOS 1 in the rat mesenteric arterial bed

Previously we have demonstrated functional nitric oxide synthase (NOS) 1 in large arteries. Because resistance arteries largely determine blood pressure, this study examined whether functional NOS 1 also exists in resistance arteries. Phenylephrine (PE) contraction was measured in the absence and presence of the NOS 1 inhibitor N(5)-(1-imino-3-butenyl)-L-ornithine (VNIO) in isolated mesenteric resistance arteries (endothelium intact and denuded) from Sprague-Dawley rats. For NOS 1 activity and expression, the mesenteric arterial bed was separated into cytosolic and particulate fractions. NOS activity was assayed by measuring the conversion of [(3)H]arginine to [(3)H]citrulline inhibited by a nonselective NOS inhibitor or VNIO. VNIO increased PE sensitivity in endothelium-intact and -denuded arteries. In cytosolic and particulate fractions of the arterial bed, approximately 40% of NOS activity was inhibited by VNIO. Immunoprecipitation and Western blot analysis revealed two NOS 1 immunoreactive bands. One band corresponded to the rat brain isoform, whereas the second was of a slightly lower molecular mass. The cytosolic fraction contained both isoforms; however, the particulate fraction had only the lower molecular mass form. These studies demonstrate the existence of functional NOS 1 in resistance arteries.     

Fatal cytauxzoonosis in a free-ranging bobcat (Lynx rufus)

In September 2000, a free-ranging bobcat (Lynx rufus) cub was presented to the Kansas State University Veterinary Teaching Hospital (Manhattan, Kansas, USA) in a moribund state with signs of severe anemia and respiratory difficulty. The cub was euthanized. Gross necropsy findings included multifocal atelectasis, splenomegaly, and pericardial effusion. Microscopic examination revealed subacute pulmonary thrombosis, mild vasculitis in the brain, and large schizont-filled macrophages within blood vessels of all tissues examined. The organisms were typical of the developmental stages of Cytauxzoon felis. Cytauxzoonosis is considered to be a persistent, subclinical infection in the bobcat; however, this cub had lesions consistent with those seen in fatal infections in domestic cats. This case of fatal C. felis infection indicates that some free-ranging bobcats may die of cytauxzoonosis.     

Field-based evaluation of biopesticide impacts on native biodiversity: malagasy coleoptera and anti-locust entomopathogenic fungi

A community of 225 species of Coleoptera was used as a surrogate to evaluate nontarget effects of entomopathogenic fungi under development as biopesticides for use against the Malagasy migratory locust Locusta migratoria capito Saussure (Orthoptera: Acrididae). Evaluation of a standard chemical treatment of fenitrothion + esfenvalerate, two indigenous isolates of Metarhiziumflavoviride Gams & Roszsypol (SP3 and SP9), and an indigenous isolate of Beauveria bassiana (Balsamo) Vuillemin (SP16) against an untreated control in a replicated field trial in southern Madagascar showed that one of the isolates of M. flavoviride (SP3) and fenitrothion + esfenvalerate had distinct effects on nontarget beetle communities that were similar to each other. The other two isolates had no detectable effects compared with the untreated control. Based on an evaluation of the species affected, the similar effects of SP3 and the chemical pesticide are hypothesized to be the result of a perturbation of predator-prey relationships, with a distinct tendency to be manifested via predators. The data indicate that use of SP9 and SP16 would have minimal detrimental effects on the biodiversity of nontarget beetles, but that SP3 needs further testing.     

Receptor-specific influence of endothelin-1 in the erectile response of the rat

Specific receptor antagonists were used to examine the role of endothelin-1 (ET-1) in the erectile response of the rat. In these studies, intact rats were cannulated to permit the continuous recording of mean arterial pressure (MAP) and intracavernosal pressure (CCP). Erection was induced by electrical stimulation of the autonomic ganglion, which regulates blood flow to the penis. The animals were subjected to intracavernosal injection with vehicle only (Cont) or with an antagonist to the endothelin-A receptor (ET(A)) or to the endothelin-B receptor (ET(B)). Blockade of the ET(A) or the ET(B) had no effect on the erectile response (CCP/MAP) during maximal ganglionic stimulation. When ET-1 was injected into Cont rats, there was a marked vasoconstriction with a sharp rise in MAP and a decline in CCP as the cavernosal arterioles constricted and limited inflow. The injection of the ET(A) antagonist prevented the vasoconstriction after ET-1 injection into Cont rats, whereas blockade of the ET(B) had no effect on the vasoconstrictive effect to ET-1. Similar results were obtained during submaximal ganglionic stimulation. With minimal levels of ganglionic stimulation, ET-1 injection led to a moderated degree of vasodilation in the presence of the ET(A) antagonist. The ET(B) antagonist failed to alter the CCP response during minimal stimulation, but it did have a marked effect on the MAP response to ET-1 injection. The results of these studies confirm that cavernosal tissue of the rat penis is highly responsive to ET-1. However, the failure of the ET-1 antagonists to affect penile erection in response to ganglionic stimulation reflects a minimal role of ET-1 in the erectile response in the rat.     

Inhibition of expression of the type I G protein-coupled receptor for vasoactive intestinal peptide (VPAC1) by hammerhead ribozymes

Vasoactive intestinal peptide (VIP) is a neuromediator expressed widely in the nervous, gastrointestinal, respiratory, and immune systems. Two G protein-coupled receptors (GPCRs), designated VPAC1 and VPAC2, bind VIP with high affinity and transduce increases in [cyclic AMP](i) and [Ca(2+)](i). As there are no potent VPAC1- or VPAC2-selective antagonists, a hammerhead ribozyme (Rz) strategy capable of in vivo application was adopted to inactivate individual domains of VPAC1. Three Rzs were designed to cleave mRNA encoding the amino terminus, the third intracellular loop, and the cytoplasmic tail of human VPAC1 and were introduced by transfection into HEK-293 cells expressing recombinant human VPAC1. Each Rz specifically degraded VPAC1 mRNA and down-regulated VPAC1 protein and VIP-binding activity, as assessed by ribonuclease protection assays, Western blots, and binding of (125)I-VIP. Rz-mediated down-regulation of VPAC1 was associated with up to 75% suppression of VIP signaling of increases in [cyclic AMP](i) and [IP3](i), and of cyclic AMP response element-luciferase reports. The Rz specific for the amino terminus inhibited VPAC1 expression and signaling to the greatest extent. VIP-evoked cellular responses thus appear to be proportional to the level of VPAC1 expression. Specific Rzs may be powerful tools for manipulating tissue-specific contributions of GPCRs in vitro and in vivo.     

Children have less variable postures and muscle activities when using new electronic information technology compared with old paper-based information technology

Children now have considerable exposure to new information technologies (IT) such as desktop computers. A reported association between computer use and discomfort in children has prompted concerns about the musculoskeletal stresses associated with computer use. There were no detailed data on children reading and writing, nor any evidence on the variability of postures and muscle activity whilst children use IT.Twenty-four children (10–12 years old; 12 male) performed a reading and writing task using new IT (computer/keyboard/mouse with high display and mid height display) and old IT (book/paper/pen). Spinal and upper limb 3D posture and muscle activity were recorded and estimates of mean and variation calculated.The mean postures for children reading and writing with computers were more neutral than when they read and wrote with old IT. Similarly, mean muscle activity levels were lower during computer use than old IT use. However, new IT use also resulted in less variable, more monotonous postures and muscle activities. Moderate differences in computer display height had little effect on posture and muscle activity variation.Variation in musculoskeletal stresses is considered an important component of the risk of musculoskeletal disorders. Children should therefore be encouraged to ensure task variety when using new IT to offset the greater posture and muscle activity monotony.     

Open Capture–Recapture Models with Heterogeneity: II. Jolly–Seber Model

Summary Estimation of abundance is important in both open and closed population capture–recapture analysis, but unmodeled heterogeneity of capture probability leads to negative bias in abundance estimates. This article defines and develops a suite of open population capture–recapture models using finite mixtures to model heterogeneity of capture and survival probabilities. Model comparisons and parameter estimation use likelihood‐based methods. A real example is analyzed, and simulations are used to check the main features of the heterogeneous models, especially the quality of estimation of abundance, survival, recruitment, and turnover. The two major advances in this article are the provision of realistic abundance estimates that take account of heterogenetiy of capture, and an appraisal of the amount of overestimation of survival arising from conditioning on the first capture when heterogeneity of survival is present.     

Structural relatedness between mosquitocidal endotoxins of Bacillus thuringiensis subsp. israelensis

A mosquitocidal toxin gene, cloned from Bacillus thuringiensis subsp. israelensis, was introduced into mutant crystal-negative B. thuringiensis subsp. israelensis cells. Partial toxicity to mosquitos was restored. The 58-kilodalton cloned gene product is a minor protein component of B. thuringiensis subsp. israelensis crystals and is structurally related to a major, 135-kilodalton crystal toxin.