Structural basis for the inhibition of firefly luciferase by a general anesthetic.

The firefly luciferase enzyme from Photinus pyralis is probably the best-characterized model system for studying anesthetic-protein interactions. It binds a diverse range of general anesthetics over a large potency range, displays a sensitivity to anesthetics that is very similar to that found in animals, and has an anesthetic sensitivity that can be modulated by one of its substrates (ATP). In this paper we describe the properties of bromoform acting as a general anesthetic (in Rana temporaria tadpoles) and as an inhibitor of the firefly luciferase enzyme at high and low ATP concentrations. In addition, we describe the crystal structure of the low-ATP form of the luciferase enzyme in the presence of bromoform at 2.2-A resolution. These results provide a structural basis for understanding the anesthetic inhibition of the enzyme, as well as an explanation for the ATP modulation of its anesthetic sensitivity.     

Mapping the polarity profiles of general anesthetic target sites using n-alkane-(alpha, omega)-diols

The effects of the homologous series of n-alkane-(alpha, omega)-diols have been studied on the inhibition of the purified firefly luciferase enzyme from Photinus pyralis, the inhibition of the purified bacterial luciferase enzyme from Vibrio harveyi, and the induction of general anesthesia in Xenopus laevis tadpoles. All but one of the diols tested were found to be reversible general anesthetics. The diols inhibited firefly luciferase by competing with its normal substrate firefly luciferin, and they inhibited bacterial luciferase by competing with the substrate n-decanal. For all but the smallest agent (1,4-butanediol), only a single diol molecule was found to be involved in the inhibition of the enzymes. Inhibition constants Ki were determined for the enzymes, and general anesthetic EC50 concentrations were determined for tadpoles. These data were then used in conjunction with previously determined n-alkane and n-alcohol data to calculate, as a function of chain length, the incremental standard Gibbs free energies delta (delta G0) for adding apolar -CH2- groups and for converting apolar terminal -CH3 groups to polar -CH2OH groups. The resulting plots of delta (delta G0) versus chain length gave a consistent mapping of the polarity profiles of the anesthetic-binding pockets. They clearly reveal the existence of two substantial and distinct polar regions in the anesthetic-binding pocket of firefly luciferase but only one such region for bacterial luciferase and for the unknown target sites underlying general anesthesia. The polarities and geometric properties of these different binding sites for straight-chain anesthetics are discussed in terms of simple models.     

Dynamics of firefly luciferase inhibition by general anesthetics: Gaussian and anisotropic network analyses

The new crystal structures of the product-bound firefly luciferase combined with the previously determined substrate-free structures allow for a detailed analysis of the dynamics basis for the luciferase enzymatic activities. Using the Gaussian network model and the anisotropic network model, we show here that the superposition of the three slowest anisotropic network model modes, consisting of the bending, rotating, and rocking motions of the C-domain, accounts for large rearrangement of domains from the substrate-free (open) to product-bound (closed) conformation and thus constitutes a critical component of the enzyme's functions. The analysis also offers a unique platform to reexamine the molecular mechanism of the anesthetic inhibition of the firefly luciferase. Through perturbing the protein backbone network by introducing additional nodes to represent anesthetics, we found that the presence of two representative anesthetics, halothane and n-decanol, in different regions of luciferase had distinctively different effects on the protein's global motion. Only at the interface of the C- and N-domains did the anesthetics cause the most profound reduction in the overall flexibility of the C-domain and the concomitant increase in the flexibility of the loop, where the substitution of a conserved lysine residue was found experimentally to lead to >2-3 orders of magnitude reduction in activity. These anesthetic-induced dynamics changes can alter the normal function of the protein, appearing as an epiphenomenon of an "inhibition". The implication of the study is that a leading element for general anesthetic action on proteins is to disrupt the modes of motion essential to protein functions.     

Exploring Volatile General Anesthetic Binding to a Closed Membrane-Bound Bacterial Voltage-Gated Sodium Channel via Computation

Despite the clinical ubiquity of anesthesia, the molecular basis of anesthetic action is poorly understood. Amongst the many molecular targets proposed to contribute to anesthetic effects, the voltage gated sodium channels (VGSCs) should also be considered relevant, as they have been shown to be sensitive to all general anesthetics tested thus far. However, binding sites for VGSCs have not been identified. Moreover, the mechanism of inhibition is still largely unknown. The recently reported atomic structures of several members of the bacterial VGSC family offer the opportunity to shed light on the mechanism of action of anesthetics on these important ion channels. To this end, we have performed a molecular dynamics “flooding” simulation on a membrane-bound structural model of the archetypal bacterial VGSC, NaChBac in a closed pore conformation. This computation allowed us to identify binding sites and access pathways for the commonly used volatile general anesthetic, isoflurane. Three sites have been characterized with binding affinities in a physiologically relevant range. Interestingly, one of the most favorable sites is in the pore of the channel, suggesting that the binding sites of local and general anesthetics may overlap. Surprisingly, even though the activation gate of the channel is closed, and therefore the pore and the aqueous compartment at the intracellular side are disconnected, we observe binding of isoflurane in the central cavity. Several sampled association and dissociation events in the central cavity provide consistent support to the hypothesis that the “fenestrations” present in the membrane-embedded region of the channel act as the long-hypothesized hydrophobic drug access pathway.     

A Unitary Anesthetic Binding Site at High Resolution

Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABA_A receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABA_A receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show that apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABA_A receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels.Most general anesthetics alter the activity of ligand-gated ion channels, and electrophysiology, photolabeling, and transgenic animal experiments imply that this effect contributes to the mechanism of anesthesia (1–9). Although the molecular mechanism for this effect is not yet clear, photolabeling studies indicate that anesthetics bind within the transmembrane regions of Cys-loop ligand-gated ion channels such as the nicotinic acetylcholine and the γ-aminobutyric acid (GABA)² type A receptors (2, 9–11). Practical difficulties associated with overexpression, purification, and crystallization of ion channels have thus far stymied investigation of the structural and energetic bases underlying anesthetic recognition. However, general anesthetics also bind specifically to sites in soluble proteins, including firefly luciferase, human serum albumin (HSA), and horse spleen apoferritin (HSAF) (12–14), and x-ray crystal structures have been determined for complexes of these proteins with several general anesthetics (14–16). In particular, HSAF is an attractive model for studying anesthetic-protein interactions because it has the highest affinity for anesthetics of any protein studied to date, has a unique anesthetic binding site, and is a multimer of 4-helix bundles, much like the putative anesthetic binding regions in ligand-gated channels. In addition, apoferritin is commercially available and crystallizes readily. Most importantly, however, the affinity of HSAF for a broad range of general anesthetics is highly correlated with anesthetic potency, confirming the utility and relevance of this model system (17).Ferritin is a 24-mer iron-binding protein. It sequesters free iron ions, thereby helping to maintain non-toxic levels of iron in the cell and functioning as a cellular iron reservoir (18, 19). Each subunit has a molecular mass of ∼20 kDa and adopts a 4-helix bundle fold. The 24-mer forms a hollow, roughly spherical particle with 432 symmetry. Two ferritin isoforms are found in mammals, heavy (H) and light (L), and 24-mers can contain all H chains, all L chains, or mixtures of varying stoichiometry; the biological significance of the H/L ratio is not yet clear (20).In addition to the large central cavity, the apoferritin 24-mer contains additional, smaller cavities at the dimer interfaces; these smaller cavities are of an appropriate size to accommodate anesthetics. X-ray crystallography has confirmed that this interfacial cavity is the binding site for the inhalational anesthetics halothane and isoflurane, and isothermal titration calorimetry (ITC) measurements have shown that this interfacial site has a relatively high affinity for these anesthetics (K_a values ∼10⁵ m⁻¹) (14).General anesthetics fall into at least two broad classes, inhalational and injectable. Whereas both classes of drugs can induce the amnesia, immobility, and hypnosis associated with anesthesia, molecules in the two classes differ substantially in their chemical and physical properties. Prior to this work, only one crystal structure has been available for an injectable general anesthetic complexed with a protein-propofol, bound to HSA (16). This structure revealed that the propofol binding sites on this protein do not, by and large, overlap with the binding sites for inhalational anesthetics. This raises the question of whether the two types of drug invariably bind to separate sets of targets, or whether they could possibly transduce their effects by binding to a single protein site. To address this question we assessed whether propofol binds to the apoferritin site that had been previously identified as the binding site for inhalational anesthetics. Using x-ray crystallography, calorimetry, and molecular modeling, we show that the two types of anesthetics do indeed share a common binding site. We also investigated structure-binding relationships for a homologous series of propofol-like compounds and found that, remarkably, the energetics of binding to apoferritin precisely match the compound''s abilities to potentiate GABA effects at GABA_A receptors, suggesting that similar structural and physicochemical factors mediate anesthetic recognition by both apoferritin and ligand-gated ion channels. This argues for the possibility that anesthetic binding might trigger structural and dynamic alterations in GABA_A receptors similar to those observed in apoferritin, and that these changes underlie anesthetic effects.     

Cooperative binding of inhaled anesthetics and ATP to firefly luciferase

Firefly luciferase is considered a reasonable model of in vivo anesthetic targets despite being destabilized by anesthetics, as reflected by differential scanning calorimetry (DSC). We examined the interaction between two inhaled anesthetics, ATP, luciferase, and temperature, using amide hydrogen exchange, tryptophan fluorescence, and photolabeling in an attempt to examine this apparent discrepancy. In the absence of ATP/Mg2+, halothane and bromoform cause destabilization, as measured by hydrogen exchange, suggesting nonspecific interactions. In the presence of ATP/Mg2+ and at room temperature, the anesthetics produce considerable stabilization with a negative DeltaH, indicating population of a conformer with a specific anesthetic binding site. Stabilizing interactions are lost, however, at unfolding temperatures. We suggest that preferential binding to aggregated forms of luciferase explain the higher temperature destabilization detected with DSC. Our results demonstrate a cooperative binding equilibrium between native ligands and anesthetics, suggesting that similar interactions could underlie actions at biologically relevant targets.     

Propofol, a general anesthetic, promotes the formation of fluid phase domains in model membranes

The molecular site of anesthetic action remains an area of intense research interest. It is not clear whether general anesthetics act through direct binding to proteins or by perturbing the membrane properties of excitable tissues. Several studies indicate that anesthetics affect the properties of either membrane lipids or proteins. However, gaps remain in our understanding of the molecular mechanism of anesthetic action. Recent developments in membrane biology have led to the concept of small-scale domain structures in lipid and lipid--protein coupled systems. The role of such domain structures in anesthetic action has not been studied in detail. In the present study, we investigated the effect of anesthetics on lipid domain structures in model membranes using the fluorescent spectral properties of Laurdan (6-dodecanoyl-2-dimethylamino naphthalene). Propofol, a general anesthetic, promoted the formation of fluid domains in model membranes of dipalmitoyl phosphatidyl choline (DPPC) or mixtures of lipids of varying acyl chains (DPPC:DMPC dimyristoyl phosphatidyl choline 1:1). The estimated size of these domains is 20--50 A. Based on these studies, we speculate that the mechanism of anesthetic action may involve effects on protein--lipid coupled systems through alterations in small-scale lipid domain structures.     

Molecular mapping of general anesthetic sites in a voltage-gated ion channel

Several voltage-gated ion channels are modulated by clinically relevant doses of general anesthetics. However, the structural basis of this modulation is not well understood. Previous work suggested that n-alcohols and inhaled anesthetics stabilize the closed state of the Shaw2 voltage-gated (Kv) channel (K-Shaw2) by directly interacting with a discrete channel site. We hypothesize that the inhibition of K-Shaw2 channels by general anesthetics is governed by interactions between binding and effector sites involving components of the channel's activation gate. To investigate this hypothesis, we applied Ala/Val scanning mutagenesis to the S4-S5 linker and the post-PVP S6 segment, and conducted electrophysiological analysis to evaluate the energetic impact of the mutations on the inhibition of the K-Shaw2 channel by 1-butanol and halothane. These analyses identified residues that determine an apparent binding cooperativity and residue pairs that act in concert to modulate gating upon anesthetic binding. In some instances, due to their critical location, key residues also influence channel gating. Complementing these results, molecular dynamics simulations and in silico docking experiments helped us visualize possible anesthetic sites and interactions. We conclude that the inhibition of K-Shaw2 by general anesthetics results from allosteric interactions between distinct but contiguous binding and effector sites involving inter- and intrasubunit interfaces.     

NMR resolved multiple anesthetic binding sites in the TM domains of the α4β2 nAChR

The α4β2 nicotinic acetylcholine receptor (nAChR) has significant roles in nervous system function and disease. It is also a molecular target of general anesthetics. Anesthetics inhibit the α4β2 nAChR at clinically relevant concentrations, but their binding sites in α4β2 remain unclear. The recently determined NMR structures of the α4β2 nAChR transmembrane (TM) domains provide valuable frameworks for identifying the binding sites. In this study, we performed solution NMR experiments on the α4β2 TM domains in the absence and presence of halothane and ketamine. Both anesthetics were found in an intra-subunit cavity near the extracellular end of the β2 transmembrane helices, homologous to a common anesthetic binding site observed in X-ray structures of anesthetic-bound GLIC (Nury et al., [32]). Halothane, but not ketamine, was also found in cavities adjacent to the common anesthetic site at the interface of α4 and β2. In addition, both anesthetics bound to cavities near the ion selectivity filter at the intracellular end of the TM domains. Anesthetic binding induced profound changes in protein conformational exchanges. A number of residues, close to or remote from the binding sites, showed resonance signal splitting from single to double peaks, signifying that anesthetics decreased conformation exchange rates. It was also evident that anesthetics shifted population of two conformations. Altogether, the study comprehensively resolved anesthetic binding sites in the α4β2 nAChR. Furthermore, the study provided compelling experimental evidence of anesthetic-induced changes in protein dynamics, especially near regions of the hydrophobic gate and ion selectivity filter that directly regulate channel functions.     

Effect of general anesthetics on the properties of lipid membranes of various compositions

Computer simulations of four lipid membranes of different compositions, namely neat DPPC and PSM, and equimolar DPPC-cholesterol and PSM-cholesterol mixtures, are performed in the presence and absence of the general anesthetics diethylether and sevoflurane both at 1 and 600 bar. The results are analyzed in order to identify membrane properties that are potentially related to the molecular mechanism of anesthesia, namely that change in the same way in any membrane with any anesthetics, and change oppositely with increasing pressure. We find that the lateral lipid density satisfies both criteria: it is decreased by anesthetics and increased by pressure. This anesthetic-induced swelling is attributed to only those anesthetic molecules that are located close to the boundary of the apolar phase. This lateral expansion is found to lead to increased lateral mobility of the lipids, an effect often thought to be related to general anesthesia; to an increased fraction of the free volume around the outer preferred position of anesthetics; and to the decrease of the lateral pressure in the nearby range of the ester and amide groups, a region into which anesthetic molecules already cannot penetrate. All these changes are reverted by the increase of pressure. Another important finding of this study is that cholesterol has an opposite effect on the membrane properties than anesthetics, and, correspondingly, these changes are less marked in the presence of cholesterol. Therefore, changes in the membrane that can lead to general anesthesia are expected to occur in the membrane domains of low cholesterol content.     

Contributions of dipole moments, quadrupole moments, and molecular polarizabilities to the anesthetic potency of fluorobenzenes

Previous studies have emphasized the role of molecular polarizability and electric moments, especially dipole and quadrupole moments, in binding of drugs to sites of action. A recent publication of ED50s that prevent response to a noxious stimulus for eight fluorobenzenes has made it possible to compare anesthetic potency with ab initio Hartree-Fock calculations of molecular polarizability as well as dipole and quadrupole moments. Fluorobenzenes provide a stringent test of the role of electric moments in anesthetic potency because individual dipole moments range from 0 to 2.84 debye (D) while the quadrupole moment of benzene is large and negative (-30 x 10(-40) C m(2)), that of hexafluorobenzene is large and positive (30 x 10(-40) C m(2)), and that of 1,3,5-trifluorobenzene is nearly zero. We found that anesthetic potency of fluorobenzenes was not affected by the presence of either dipole or quadrupole moments. This result is surprising because fluoroalkanes and fluorocycloalkanes are most potent when half fluorinated and are usually not anesthetics when perfluorinated. The results suggest that electrostatic interactions are not important for binding of fluorobenzenes at sites of anesthetic action and that these sites are different from those that bind conventional anesthetics.     

Effects of general anesthetics on the bacterial luciferase enzyme from Vibrio harveyi: an anesthetic target site with differential sensitivity

The effects of a diverse range of 36 general anesthetics and anesthetic-like compounds on a highly purified preparation of the bacterial luciferase enzyme from Vibrio harveyi have been investigated. Under conditions where the flavin site was saturated, almost all of the anesthetics inhibited the peak enzyme activity and slowed the rate of decay. However, a small number of the more polar agents only inhibited at high concentrations, while stimulating activity at lower concentrations. The inhibition was found to be competitive in nature, with the anesthetics acting by competing for the binding of the aldehyde substrate n-decanal. The anesthetic binding site on the enzyme could accommodate only a single molecule of a large anesthetic but more than one molecule of a small anesthetic, consistent with the site having circumscribed dimensions. The homologous series of n-alcohols and n-alkanes exhibited cutoffs in inhibitory potency, but these cutoffs occurred at very different chain lengths (about C10 for the n-alkanes and C15 for the n-alcohols), mimicking similar cutoffs observed for general anesthetic potencies in animals. Binding constants determined from peak height measurements showed that the inhibitor binding site was predominantly hydrophobic (with a mean delta delta G CH2 of -5.0 kJ/mol), but fluctuations in the binding constants with chain length revealed regions in the binding site with polar characteristics. Binding constants to an intermediate form of the enzyme (intermediate II) were also determined, and these confirmed the principal features of the binding site deduced from the peak height measurements. The long-chain compounds, however, bound considerably tighter to the intermediate II form of the enzyme, and this was shown to account for the biphasic decay kinetics that were observed with these compounds. Overall, there was poor agreement between the EC50 concentrations for inhibiting the luciferase enzyme from V. harveyi and those which induce general anesthesia in animals, with bulky compounds being much less potent, and moderately long chain alcohols being much more potent, as luciferase inhibitors than as general anesthetics.     

A Genetic and Mosaic Analysis of a Locus Involved in the Anesthesia Response of Drosophila Melanogaster

We describe a genetic and behavioral analysis of several alleles of har38, a mutant with altered sensitivity to the general anesthetic halothane. We obtained a P-element-induced allele of har38 and generated several excision alleles by remobilizing the P element. The mutants narrow abdomen (na) and har85 are confirmed to be allelic to har38. Besides a decreased sensitivity to halothane, all mutant alleles of this locus cause a characteristic walking behavior in the absence of anesthetics. We have quantified this behavior using a geotaxis apparatus. Responses of the mutant alleles to different inhalational anesthetics were tested. The results strongly favor a multipathway model for the onset of anesthesia. Mosaic flies were tested for their response to halothane and checked for their abnormal walking behavior. The analysis suggests that both the behaviors are exhibited only by such mosaics as have the entire head of mutant origin. It is likely that this focus represents an element of a common pathway in the anesthetic response to several inhalational anesthetics but not all. This result is the first demonstration of regional specificity in the CNS of any animal for general anesthetic action.     

n-Dodecyl β-D-maltoside specifically competes with general anesthetics for anesthetic binding sites

We recently demonstrated that the anionic detergent sodium dodecyl sulfate (SDS) specifically interacts with the anesthetic binding site in horse spleen apoferritin, a soluble protein which models anesthetic binding sites in receptors. This raises the possibility of other detergents similarly interacting with and occluding such sites from anesthetics, thereby preventing the proper identification of novel anesthetic binding sites. n-Dodecyl β-D-maltoside (DDM) is a non-ionic detergent commonly used during protein-anesthetic studies because of its mild and non-denaturing properties. In this study, we demonstrate that SDS and DDM occupy anesthetic binding sites in the model proteins human serum albumin (HSA) and horse spleen apoferritin and thereby inhibit the binding of the general anesthetics propofol and isoflurane. DDM specifically interacts with HSA (K_d?=?40?μM) with a lower affinity than SDS (K_d?=?2?μM). DDM exerts all these effects while not perturbing the native structures of either model protein. Computational calculations corroborated the experimental results by demonstrating that the binding sites for DDM and both anesthetics on the model proteins overlapped. Collectively, our results indicate that DDM and SDS specifically interact with anesthetic binding sites and may thus prevent the identification of novel anesthetic sites. Special precaution should be taken when undertaking and interpreting results from protein-anesthetic investigations utilizing detergents like SDS and DDM.     

Pressure-induced exclusion of a local anesthetic from model and nerve membranes

Because it is well established that the anesthetic state can be reversed by pressure, a number of molecular theories that have been proposed for the mechanism of action of both local and general anesthetics can be tested by varying the pressure. Using Fourier transform infrared spectroscopy, we report here the first direct observation of the expulsion from lipid bilayers of a local anesthetic, tetracaine, by pressure. Moreover, we establish for the first time that this phenomenon is common to both model membranes and to myelinated and unmyelinated nerve membranes, vindicating the utility of model membrane systems. A distinctive feature of this behavior in model systems is that, in saturated phosphatidylcholines at high pH, expulsion only occurs in the presence of cholesterol, whose ordering effect on the acyl chains evidently assists pressure in squeezing the anesthetic out of the bilayer. This pressure-induced phenomenon may provide insight into the molecular mechanisms underlying the antagonistic effect of pressure against anesthesia.     

The location and nature of general anesthetic binding sites on the active conformation of firefly luciferase; a time resolved photolabeling study

Firefly luciferase is one of the few soluble proteins that is acted upon by a wide variety of general anesthetics and alcohols; they inhibit the ATP-driven production of light. We have used time-resolved photolabeling to locate the binding sites of alcohols during the initial light output, some 200 ms after adding ATP. The photolabel 3-azioctanol inhibited the initial light output with an IC50 of 200 μM, close to its general anesthetic potency. Photoincorporation of [(3)H]3-azioctanol into luciferase was saturable but weak. It was enhanced 200 ms after adding ATP but was negligible minutes later. Sequencing of tryptic digests by HPLC-MSMS revealed a similar conformation-dependence for photoincorporation of 3-azioctanol into Glu-313, a residue that lines the bottom of a deep cleft (vestibule) whose outer end binds luciferin. An aromatic diazirine analog of benzyl alcohol with broader side chain reactivity reported two sites. First, it photolabeled two residues in the vestibule, Ser-286 and Ile-288, both of which are implicated with Glu-313 in the conformation change accompanying activation. Second, it photolabeled two residues that contact luciferin, Ser-316 and Ser-349. Thus, time resolved photolabeling supports two mechanisms of action. First, an allosteric one, in which anesthetics bind in the vestibule displacing water molecules that are thought to be involved in light output. Second, a competitive one, in which anesthetics bind isosterically with luciferin. This work provides structural evidence that supports the competitive and allosteric actions previously characterized by kinetic studies.     

HCVIRES介导萤火虫荧光素酶翻译的双顺反子载体的构建与在小鼠体内的表达

将HCVIRES插入双报告基因海肾荧光素酶 (Rluc)基因和萤火虫荧光素酶 (Fluc)基因之间 ,建立了“依赖帽子的扫描机制”翻译表达Rluc ,HCVIRES调控Fluc翻译的双顺反子表达载体pCI Rluc HCVIRES Fluc ,通过酶切反应及转染HepG2细胞鉴定双荧光素酶瞬间表达活性等试验 ,证实获得了表达双荧光素酶的双顺反子载体 .并应用水压转染法将双顺反子表达质粒导入小鼠体内 ,在小鼠肝脏检测到高水平表达的Rluc和Fluc .该研究成功构建一种HCVIRES介导萤火虫荧光素酶基因表达的双顺反子载体 ,并在HepG2细胞及小鼠体内进行了瞬时表达 ,为进一步建立稳定评价靶向HCVIRES药物作用的细胞及小动物模型研究奠定了基础     

模式生物秀丽线虫与吸入麻醉药敏感性相关的基因

吸入麻醉药虽已在临床上广泛应用,然其分子作用机制和作用位点仍然不清楚。以秀丽线虫为模式生物在研究麻醉药的分子机制上有着众多优点,近年亦取得了一定的进展。以秀丽线虫作为模式生物时麻醉终点的选择主要有两种：使用大于临床浓度的吸入麻醉药,使秀丽线虫停止运动作为麻醉终点和使用接近临床浓度的吸入麻醉药,使秀丽线虫行动变得不协调和迟缓作为麻醉终点。这两种研究方法已经发现一些与吸入麻醉药敏感性相关的基因,如unc-79,unc-80,unc-9,unc-1,gas-1和unc-64等基因。这些基因主要表达于神经元,与神经突触、线粒体的功能有关。     

The Thermodynamics of General and Local Anesthesia

General anesthetics are known to cause depression of the freezing point of transitions in biomembranes. This is a consequence of ideal mixing of the anesthetic drugs in the membrane fluid phase and exclusion from the solid phase. Such a generic law provides physical justification of the famous Meyer-Overton rule. We show here that general anesthetics, barbiturates, and local anesthetics all display the same effect on melting transitions. Their effect is reversed by hydrostatic pressure. Thus, the thermodynamic behavior of local anesthetics is very similar to that of general anesthetics. We present a detailed thermodynamic analysis of heat capacity profiles of membranes in the presence of anesthetics. Using this analysis, we are able to describe experimentally observed calorimetric profiles and predict the anesthetic features of arbitrary molecules. In addition, we discuss the thermodynamic origin of the cutoff effect of long-chain alcohols and the additivity of the effect of general and local anesthetics.     

The Thermodynamics of General and Local Anesthesia

General anesthetics are known to cause depression of the freezing point of transitions in biomembranes. This is a consequence of ideal mixing of the anesthetic drugs in the membrane fluid phase and exclusion from the solid phase. Such a generic law provides physical justification of the famous Meyer-Overton rule. We show here that general anesthetics, barbiturates, and local anesthetics all display the same effect on melting transitions. Their effect is reversed by hydrostatic pressure. Thus, the thermodynamic behavior of local anesthetics is very similar to that of general anesthetics. We present a detailed thermodynamic analysis of heat capacity profiles of membranes in the presence of anesthetics. Using this analysis, we are able to describe experimentally observed calorimetric profiles and predict the anesthetic features of arbitrary molecules. In addition, we discuss the thermodynamic origin of the cutoff effect of long-chain alcohols and the additivity of the effect of general and local anesthetics.