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Membrane-associated proteins and protein complexes account for approximately a third or more of the proteins in the cell (1, 2). These complexes mediate essential cellular processes; including signal transduc-tion, transport, recognition, bioenergetics and cell-cell communication. In general, membrane proteins are challenging to study because of their insolubility and tendency to aggregate when removed from their protein lipid bilayer environment. This chapter is focused on describing a novel method for producing and solubilizing membrane proteins that can be easily adapted to high-throughput expression screening. This process is based on cell-free transcription and translation technology coupled with nanolipoprotein par ticles (NLPs), which are lipid bilayers confined within a ring of amphipathic protein of defined diameter. The NLPs act as a platform for inserting, solubilizing and characterizing functional membrane proteins. NLP component proteins (apolipoproteins), as well as membrane proteins can be produced by either traditional cell-based or as discussed here, cell-free expression methodologies.

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Delta1-49 apolipoprotein A-I fragment (Delta49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR --> M transition. Importantly the functional bR was solubilized in discoidal bR.NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Delta49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies.

We report a cell-free approach for expressing and inserting integral membrane proteins into water-soluble particles composed of discoidal apolipoprotein-lipid bilayers. Proteins are inserted into the particles, circumventing the need of extracting and reconstituting the product into membrane vesicles. Moreover, the planar nature of the membrane support makes the protein freely accessible from both sides of the lipid bilayer. Complexes are successfully purified by means of the apoplipoprotein component or by the carrier protein. The method significantly enhances the solubility of a variety of membrane proteins with different functional roles and topologies. Analytical assays for a subset of model membrane proteins indicate that proteins are correctly folded and active. The approach provides a platform amenable to high-throughput structural and functional characterization of a variety of traditionally intractable drug targets.

In this study, we compared the effect of isoflurane and desflurane on the posterior tibial somatosensory evoked potential recorded by scalp electrodes during correction of idiopathic scoliosis in pediatric patients. Depth of sedation was controlled by maintaining bispectral index (BIS) at 60 throughout the study. Comparison of patients breathing desflurane and isoflurane showed an evoked cortical amplitude (N37-P45) of 0.53 +/- 0.3 microV versus 1.3 +/- 0.8 microV (P = 0.014), respectively. In addition to this comparison, a crossover design was included whereby the desflurane or isoflurane received in the first part of the study was changed to the other anesthetic. Substituting one anesthetic for another confirmed our initial finding that the cortical evoked amplitude is greater with isoflurane than with desflurane. No differential effect was found between desflurane and isoflurane on the evoked subcortical (N31-P34) amplitude or the P37 latency.

Five components have thus far been identified that are necessary for the incorporation of selenocysteine (Sec) into approximately 25 mammalian proteins. Two of these are cis sequences, a SECIS element in the 3'-untranslated region and a Sec codon (UGA) in the coding region. The three known trans-acting factors are a Sec-specific translation elongation factor (eEFSec), the Sec-tRNA(Sec), and a SECIS-binding protein, SBP2. Here we describe a system in which the efficiency of Sec incorporation was determined quantitatively both in vitro and in transfected cells, and in which the contribution of each of the known factors is examined. The efficiency of Sec incorporation into a luciferase reporter system in vitro is maximally 5-8%, which is 6-10 times higher than that in transfected rat hepatoma cells, McArdle 7777. In contrast, the efficiency of Sec incorporation into selenoprotein P in vitro is approximately 40%, suggesting that as yet unidentified cis-elements may regulate differential selenoprotein expression. In addition, we have found that SBP2 is the only limiting factor in rabbit reticulocyte lysate but not in transfected rat hepatoma cells where SBP2 is found to be mostly if not entirely cytoplasmic despite having a strong putative nuclear localization signal. The significance of these findings with regard to the function of known Sec incorporation factors is discussed.

Background: A synergistic effect has been described when rocuronium (Roc) and mivacurium (Miv) are combined in equal (i.e. 1:1) ED95 proportions at various total doses. We have investigated the effect of Roc or Miv alone and four different ratios (1:4, 2:3, 3:2 and 4:1) of Roc and Miv mixed to a total dose of 1.33 x ED95. The primary outcome is the ratio producing the maximum enhancement of duration of clinical effect., Methods: Sixty-eight healthy children were anaesthetized with propofol, nitrous oxide and fentanyl. They then randomly received either Roc 0.4 (mg.kg(-1)), Miv 0.133 (mg.kg(-1)) or one of four Roc + Miv combinations (mg.kg(-1)): Roc 0.32 + Miv 0.027; Roc 0.24 + Miv 0.053; Roc 0.16 + Miv 0.08; and Roc 0.08 + Miv 0.106. The mechanical response of the adductor pollicis muscle to supramaximal stimulation of the ulnar nerve at the wrist was recorded., Results: Duration of effect was greater in the combination groups than that predicted from the duration of Roc or Miv used alone. Duration was maximally increased around a 1:1 ratio (2:3 and 3:2) of Roc and Miv. The likelihood of achieving 100% block was greater in combination groups compared with Roc or Miv used alone., Conclusions: Combinations of Roc and Miv show a synergistic effect, which appears maximal as the mixture approaches a 1:1 ratio of their ED95s. This combination acted as if a larger effective dose of a single (new) drug had been given, but did not offer the advantage of both rapid onset and short duration of effect.

Background: Opioids are frequently used for sedation in the Paediatric Intensive Care Unit (PICU). With time the dosing often increases because of tolerance. On cessation of the sedation there is a risk of the opioid withdrawal syndrome. The aim of our study was to evaluate methadone dosing as a risk factor for opioid withdrawal and to determine optimal dose and efficacy of methadone to prevent withdrawal., Method: We undertook a clinical, retrospective, chart review study. Data were analysed from the quality improvement initiative database of a tertiary-care 18 bed PICU., Results: Data from 30 children who received an opioid infusion for >/=7 days and subsequently received methadone for opioid withdrawal (between January 2000 and July 2001) were analysed. Nurses documented the presence or absence of withdrawal signs daily. Our unit protocol has recommended converting the patient's opioid dose into fentanyl equivalents and a dose of methadone equal to the total daily dose of fentanyl to be given three times a day. Twenty patients had no or minimal withdrawal symptoms and 10 experienced significant withdrawal. Age, weight, PRISM score, lorazepam dose, muscle relaxant use and fentanyl dose were not statistically significantly between these groups. Receiver Operator Characteristics analysis showed that 80% of the suggested methadone dose was effective in minimizing withdrawal symptoms. The odds ratio for withdrawal with <80% of the predicted methadone dose was 21., Conclusions: Inadequate methadone is a risk factor for opioid withdrawal. A daily starting methadone dose equivalent to 2.5 times the daily fentanyl dose is effective in minimizing withdrawal symptoms.

Bothropstoxin-I (BthTX-I), a myotoxic Lys49 phospholipase A(2) (PLA(2)) homologue isolated from Bothrops jararacussu snake venom, causes a range of biological effects, including myonecrosis, mouse paw edema, irreversible neuromuscular blockade and lysis of cell cultures. Among eight divalent cations assayed, Mn(2+) was the most effective in reducing mouse paw edema induced by BthTX-I (25 microg). Preincubating BthTX-I with Mn(2+) (1.0mM) reduced mouse paw edema by 70% and myotoxicity by 60% in mice injected i.m. with 50 microg toxin. Mn(2+) (50 microl of a 1mM solution) administered within 1min at the site of toxin injection was still but less effective in antagonising BthTX-I-induced myotoxicity. Mn(2+) (1.0mM) completely prevented BthTX-I (1.4 microM)-induced neuromuscular blockade in the mouse phrenic-nerve diaphragm preparation. Mn(2+) (0.25mM) protected about 85% of NB41A3 cells from lysis when coincubated with BthTX-I (1.0 microM) for 25h. Preincubation with 0.25mM Mn(2+) increased the sensitivity of the cells to subsequent lysis by BthTX-I in the absence of Mn(2+). BthTX-I (1 microM) caused extensive fatty acid release (from 0.8% of the total radiolabeled lipid in control cells to 56% with toxin) when incubated with the NB41A3 cell line for 25h. PLA(2) activity observed in cell cultures after addition of BthTX-I was considerably reduced by 0.25mM Mn(2+). Mn(2+) ions constitute a promising agent to assess the action mechanism and the effects of enzymatic inhibition on the pharmacological activity of Lys49 PLA(2) homologues.

Although well recognized for its anti-inflammatory effect on gene expression in stimulated monocytes and macrophages, IL-4 is a pleiotropic cytokine that has also been shown to enhance TNF-alpha and IL-12 production in response to stimulation with LPS. In the present study we expand these prior studies in three areas. First, the potentiating effect of IL-4 pretreatment is both stimulus and gene selective. Pretreatment of mouse macrophages with IL-4 for a minimum of 6 h produces a 2- to 4-fold enhancement of LPS-induced expression of several cytokines and chemokines, including TNF-alpha, IL-1alpha, macrophage-inflammatory protein-2, and KC, but inhibits the production of IL-12p40. In addition, the production of TNF-alpha by macrophages stimulated with IFN-gamma and IL-2 is inhibited by IL-4 pretreatment, while responses to both LPS and dsRNA are enhanced. Second, the ability of IL-4 to potentiate LPS-stimulated cytokine production appears to require new IL-4-stimulated gene expression, because it is time dependent, requires the activation of STAT6, and is blocked by the reversible protein synthesis inhibitor cycloheximide during the IL-4 pretreatment period. Finally, IL-4-mediated potentiation of TNF-alpha production involves specific enhancement of mRNA translation. Although TNF-alpha protein is increased in IL-4-pretreated cells, the level of mRNA remains unchanged. Furthermore, LPS-stimulated TNF-alpha mRNA is selectively enriched in actively translating large polyribosomes in IL-4-pretreated cells compared with cells stimulated with LPS alone.

The decoding of UGA as a selenocysteine (Sec) codon in mammalian selenoprotein mRNAs requires a selenocysteine insertion sequence (SECIS) element in the 3' untranslated region. The SECIS is a hairpin structure that contains a non-Watson-Crick base-pair quartet with a conserved G.A/A.G tandem in the core of the upper helix. Another essential component of the Sec insertion machinery is SECIS-binding protein 2 (SBP2). In this study, we define the binding site of SBP2 on six different SECIS RNAs using enzymatic and hydroxyl radical footprinting, gel mobility shift analysis, and phosphate-ethylation binding interference. We show that SBP2 binds to a variety of mammalian SECIS elements with similar affinity and that the SBP2 binding site is conserved across species. Based on footprinting studies, SBP2 protects the proximal part of the hairpin and both strands of the lower half of the upper helix that contains the non-Watson-Crick base pair quartet. Gel mobility shift assays showed that the G.A/A.G tandem and internal loop are critical for the binding of SBP2. Modification of phosphates by ethylnitrosourea along both strands of the non-Watson-Crick base pair quartet, on the 5' strand of the lower helix and part of the 5' strand of the internal loop, prevented binding of SBP2. We propose a model in which SBP2 covers the central part of the SECIS RNA, binding to the non-Watson-Crick base pair quartet and to the 5' strands of the lower helix and internal loop. Our results suggest that the affinity of SBP2 for different SECIS elements is not responsible for the hierarchy of selenoprotein expression that is observed in vivo.

Background: Malignant hyperthermia (MH) is a disorder of skeletal muscle manifested as a life-threatening hypermetabolic crisis in susceptible individuals after exposure to inhalational anesthetics and depolarizing muscle relaxants. Mutations in the gene encoding the skeletal muscle ryanodine receptor (RYR1) are considered a common cause of the disorder, and, to date, more than 20 RYR1 mutations have been reported in European and Canadian families. Some studies suggest that differences may exist in the frequencies and distribution of mutations in the RYR1 gene between European and North American MH families the frequency and distribution of mutations in the RYR1 gene., Methods: Skeletal muscle samples from 73 unrelated individuals diagnosed as MH susceptible according to the North American MH caffeine-halothane contracture test were studied. Genomic DNA of MH-susceptible patients was investigated by polymerase chain reaction-based restriction fragment length polymorphism, single-strand conformation polymorphism, and sequencing analysis. The majority of known RYR1 mutations were analyzed using the restriction fragment length polymorphism method, whereas new mutations were searched by single-strand conformation polymorphism in exons 12, 15, 39, 40, 44, 45, and 46 of the gene., Results: Seven known RYR1 mutations (Arg163Cys, Gly248Arg, Arg614Cys, Val2168Met, Thr2206Met, Gly2434Arg, and Arg2454His) were detected at frequencies of 2.7, 1.4, 1.4, 1.4, 1.4, 5.5, and 4.1%, respectively. In addition, three novel amino acid substitutions (Val2214Ile, Ala2367Thr, and Asp2431Asn) were detected at frequency of 1.4% each. These 10 mutations account for 21.9% of the North American MH-susceptible population., Conclusion: Three novel candidate mutations in the RYR1 gene were identified in these MH patients. The frequency and distribution of RYR1 mutations observed in this North American MH population was markedly different from that previously identified in Europe. Larger-scale studies are necessary to clarify the type and frequency of mutations in RYR1 associated with MH in North American families.

Objective: To determine the incidence of physiologic deterioration in critically ill and injured pediatric patients during interhospital transport with air and ground ambulance, Design: Prospective, descriptive study, Setting: All children were treated in regional hospitals and then transported to a pediatric tertiary care center., Patients: Children (n = 100) with a median age of 1.4 years (range 1 week to 18 years), Main Results: Three sets of physiologic scores were calculated: at the time of referral, on departure from the referring hospital, and arrival at the tertiary care center. The incidence of significant physiologic deterioration based on the calculated physiologic scores was 5.6% (n = 4) during ground and 3.4% (n = 1) during air ambulance transports. Critical events occurred in 15% of ground and 31% of air ambulance transports., Conclusion: No difference existed in the incidence of adverse events or physiologic deterioration when air ambulance transports were compared with ground ambulance transports for critically ill children by our team. The physiologic scoring system we chose is simple and easy to use for quality assurance.

Objective: To document the safety and efficacy of an anaesthetic technique in paediatric patients undergoing transoesophageal echocardiography (TOE)., Methods: Prospective descriptive study performed in a children's hospital with all patients undergoing TOE. Topical analgesia of the pharynx was achieved with lidocaine. Anaesthesia was induced with midazolam (25 microg.kg-1), fentanyl (1 microg.kg-1), and propofol (0.5-1 mg.kg-1), followed by a continuous infusion of propofol (5-10 mg.kg-1.h-1)., Results: Thirty patients are reported. The mean age was 11.4 +/- 5.1 years (range 1-22) and weight 40.5 +/- 22.1 kg (range 10-110). All the patients tolerated the procedure well. Two patients experienced brief oxygen desaturations during induction, 10 patients coughed during the procedure, and six patients had significant muscle activity requiring supplemental doses of propofol. None of the patients experienced nausea or vomiting., Conclusion: We conclude that our anaesthetic technique in spontaneously breathing paediatric patients during TOE is effective and appears to be safe in children with heart disease.

The translation of mammalian selenoprotein mRNAs requires the 3' untranslated region that contains a selenocysteine insertion sequence (SECIS) element necessary for decoding an in-frame UGA codon as selenocysteine (Sec). Selenoprotein biosynthesis is inefficient, which may be due to competition between Sec insertion and termination at the UGA/Sec codon. We analyzed the polysome distribution of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, a member of the glutathione peroxidase family of selenoproteins, in rat hepatoma cell and mouse liver extracts. In linear sucrose gradients, the sedimentation velocity of PHGPx mRNA was impeded compared to CuZn superoxide dismutase (SOD) mRNA, which has a coding region of similar size. Selenium supplementation increased the loading of ribosomes onto PHGPx mRNA, but not CuZn SOD mRNA. To determine whether the slow sedimentation velocity of PHGPx mRNA is due to a block in elongation, we analyzed the polysome distribution of wild-type and mutant mRNAs translated in vitro. Mutation of the UGA/Sec codon to UGU/cysteine increased ribosome loading and protein synthesis. When UGA/Sec was replaced with UAA or when the SECIS element core was deleted, the distribution of the mutant mRNAs was similar to the wild-type mRNA. Addition of SECIS-binding protein SBP2, which is essential for Sec insertion, increased ribosome loading and translation of wild-type PHGPx mRNA, but had no effect on the mutant mRNAs. These results suggest that elongation is impeded at UGA/Sec, and that selenium and SBP2 alleviate this block by promoting Sec incorporation instead of termination.

Objective: To determine sensitivity of equine skeletal muscle to tetrodotoxin and compare that with sensitivity of murine and human skeletal muscles., Sample Population: Semimembranosus, vastus lateralis, triceps brachii, and masseter muscle specimens from 22 euthanatized horses, vastus lateralis muscle biopsy specimens from 25 clinically normal humans, and diaphragmatic muscle specimens from 6 mice., Procedure: Electrically elicited twitch responses were measured in muscle specimens incubated in medium alone and with tetrodotoxin (100 nM, 400 nM, 1.6 microM for equine specimens and 100 nM, 200 nM, 400 nM, 800 nM, 1.6 microM for murine and human specimens). Percentages of tetrodotoxin-sensitive and -resistant sodium channels were determined and compared among muscles and species., Results: 2 sodium channels with different sensitivities to tetrodotoxin were identified in equine muscle. One was blocked with 100 nM tetrodotoxin and the other was unaffected by tetrodotoxin at concentrations up to 1.6 microM. The only difference detected among the 4 equine muscles was that masseter muscle specimens had a higher percentage of tetrodotoxin-sensitive channels than triceps brachii muscle specimens. Tetrodotoxin-resistant sodium channels constituted 31 to 66% of the equine muscle twitch response, which was greater than that determined for normal human and murine muscle (< 5%)., Conclusion and Clinical Relevance: Equine skeletal muscle contains a high percentage of tetrodotoxin-resistant sodium channels. The 4 equine muscles evaluated were more similar to each other than to murine and human muscles. Shifts in expression of sodium channel subtypes may play a role in the manifestation of certain myopathies.

In eukaryotes, the decoding of the UGA codon as selenocysteine (Sec) requires a Sec insertion sequence (SECIS) element in the 3' untranslated region of the mRNA. We purified a SECIS binding protein, SBP2, and obtained a cDNA clone that encodes this activity. SBP2 is a novel protein containing a putative RNA binding domain found in ribosomal proteins and a yeast suppressor of translation termination. By UV cross-linking and immunoprecipitation, we show that SBP2 specifically binds selenoprotein mRNAs both in vitro and in vivo. Using (75)Se-labeled Sec-tRNA(Sec), we developed an in vitro system for analyzing Sec incorporation in which the translation of a selenoprotein mRNA was both SBP2 and SECIS element dependent. Immunodepletion of SBP2 from the lysates abolished Sec insertion, which was restored when recombinant SBP2 was added to the reaction. These results establish that SBP2 is essential for the co-translational insertion of Sec into selenoproteins. We hypothesize that the binding activity of SBP2 may be involved in preventing termination at the UGA/Sec codon.

We have purified a cytotoxic L-amino acid oxidase (LAO) from Agkistrodon contortrix laticinctus snake venom by means of Superdex-200 gel filtration, followed by phenyl-Sepharose CL-4B chromatography. The purified enzyme (ACL LAO) is a dimer on gel filtration, with a M(r) of 60,000 for the monomer as estimated by SDS-PAGE. LAO activity was tested against 15 amino acids, but only 9 were oxidized by the enzyme, suggesting that it presents some degree of specificity. ACL LAO has apoptosis-inducing activity in an HL-60 cell culture assay. After 24 h treatment with 25 micrograms/ml of ACL LAO, the typical DNA fragmentation pattern of apoptotic cells was observed on agarose gel electrophoresis. NMR analysis showed the presence of a flavin mononucleotide prosthetic group. To solve its 3-D structure, crystals of the purified protein were grown in 0.1 M Tris-HCl, pH 8.5, and 2 M (NH(4))(2)SO(4). Diffraction data collected to 3.5 A showed that the protein crystallized in the tetragonal system, with unit cell a = b = 103.22 A, c = 183.45 A. This is the first report of preliminary crystallization data for a snake venom L-amino acid oxidase., (Copyright 1999 Academic Press.)

Background: The function or expression of subtypes of the sodium ion (Na+) channel is altered in biopsies or cultures of skeletal muscle from many persons who are susceptible to malignant hyperthermia (MH). ATX II, a specific Na+ channel toxin from a sea anemone, causes delayed inactivation of the channel similar to that seen in cell cultures of MH muscle. ATX II was added to skeletal muscle to determine whether altered Na+ channel function could increase the sensitivity of normal skeletal muscle to agents (halothane, caffeine, ryanodine) to which MH muscle is hypersensitive., Methods: Studies were performed of fiber bundles from the vastus lateralis muscle of persons who were deemed not MH susceptible (MH-) or MH susceptible (MH+) according to the MH diagnostic test and of strips of diaphragm muscle from rats. Preparations in a tissue bath containing Krebs solution were connected to a force transducer. ATX II was introduced 5 min before halothane, caffeine, or ryanodine., Results: ATX II increased the magnitude of contracture to halothane in preparations from most MH-, but not MH+, human participants. After ATX II treatment, preparations from 9 of 24 MH- participants generated contractures to halothane, 3%, that were of the same magnitude as those from MH+ participants. Preparations from four of six ATX II-treated healthy participants also gave responses of the same magnitude as those of MH-susceptible participants to a graded halothane challenge (0.5-3%). The contractures to bolus doses of halothane in specimens from male participants were more than three times larger than the contractures in specimens from female participants. In rat muscle, ATX II increased the magnitude of contracture to caffeine (2 mM) and decreased the time to produce a 1-g contracture to ryanodine (1 microM)., Conclusions: ATX II, which causes delayed inactivation of the Na+ channel in cell cultures similar to that reported in cultures of MH+ skeletal muscle, increased the sensitivity of normal muscle to three agents to which MH+ muscle is hypersensitive. The increased sensitivity to halothane, 3%, occurred in most (79%), but not all, MH- participants, and this effect was most evident in male participants. Therefore, abnormal function of the Na+ channel, even if it is a secondary event in MH, may contribute to a positive contracture test result for MH.