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The enterovirus genus encompasses many clinically important human pathogens such as poliovirus, coxsackieviruses, echoviruses, numbered enteroviruses and rhinoviruses. These viruses are the etiological agents of several human diseases, including hand-foot-and-mouth disease, neonatal sepsis, encephalitis, meningitis, paralysis and respiratory infections. There is an unmet need for antivirals to treat these diseases. The non-structural protein 2C is a AAA+ helicase and plays a key role in viral replication. As such, it is an attractive target for antiviral drug development. Several repurposing screens with FDA-approved drugs have identified 2C-targeting compounds such as fluoxetine and dibucaine, but the molecular basis of 2C inhibition has remained enigmatic. Here we present the 1.5 Å resolution crystal structure of the soluble fragment of coxsackievirus B3 2C protein in complex with (S)-fluoxetine (SFX), which reveals a conserved, hydrophobic drug-binding pocket which is distal to the ATP binding site. To decipher the molecular mechanism of inhibition by fluoxetine and other 2C-targeting compounds, we engineered a soluble, hexameric and ATPase competent 2C protein. Using this system, we show that SFX, dibucaine, HBB and guanidine hydrochloride inhibit 2C ATPase activity in a dose-dependent manner. Moreover, using cryo-EM analysis, we demonstrate that SFX and dibucaine lock 2C in a defined hexameric state, rationalizing their mode of inhibition and allowing us to generate the first reconstruction of the oligomeric complex. Taken together, these results provide important structural and mechanistic insights into 2C inhibition and provide a robust engineering strategy which can be used for structural, functional and drug-screening analysis of 2C proteins from current or future enteroviruses.

Electrochemical characterization and detection of protonated dibucaine (DIC+) at microinterface array across water|1,6-dichlorohexane were performed using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Some thermodynamic parameters of dibucaine, such as the standard transfer potential, the Gibbs energy of transfer and the partition coefficient, were estimated by CV. In addition to the analytical parameters, the impact of bovine serum albumin (BSA) on dibucaine detection (in artificial serum matrices) was also investigated. DPV was applied to detect a lower concentration of DIC+, resulting in a detection limit of 0.9 &plusmn, 0.06 &micro, M. While the presence of BSA affected CV, demonstrated as reduced current responses, DPV was confirmed to be an efficient method for lowering concentrations of the dibucaine detection in the artificial serum matrix in the presence of BSA, with a limit of detection (LOD) of 1.9 &plusmn, 0.12 &micro, M.

To investigate the relationship between acetyl cholinesterase associated collagen gene (COLQ) mutation in patients with acetyl cholinesterase deficiency and its clinical characteristics. Serum and red blood cell acetyl cholinesterase from patients with acetyl cholinesterase deficiency (n=6) and normal controls (n=20) were measured by butyryl thiocholine substrate. COLQ gene variations were detected by sequencing. And the cholinesterase (ChE) genotypes were measured by dibucaine inhibition in vitro. The distributions of ChE surrounded the blood vessels and nerve fibers in lung or pancreas tissues were detected by immunohistochemical staining and indirect immunofluorescence. Serum lactic acid, ammonia and other clinical data were analyzed. Serum ChE in patients with acetyl cholinesterase deficiency were only 1/50 to 1/1000 fold of normal controls. Comparing to controls, dibucaine inhibition values of patients were significantly lower, while there were no differences in red blood cells acetyl cholinesterase. Serum lactic acid and ammonia in patients were significantly higher than controls. Inser 1281-1282 GC of COLQ gene was found in 2 patients, while IVS 6 + 21 T > A, IVS 6 + 30 G > T, IVS 6 + 34 T > C and IVS66 + 12 inser T mutations were found in the other 4 patients, respectively. In addition, the patients with COLQ gene mutation were resistant to regular doses of anesthetics. COLQ gene mutation may be an important reason for the lack of serum ChE in patients with acetyl cholinesterase deficiency.

Paraquat (PQ) as a Parkinsonian mimetic has been demonstrated to impair dopaminergic (DAergic) neurons and is highly correlated with the etiology of Parkinson's disease (PD) where the death of DAergic neurons has been mainly attributed to impaired mitochondrial functioning. In this study, PQ-induced cytotoxicity focusing on mitochondrial membrane permeability (MMP), which has been implicated to play a part in neurodegeneration, was investigated. Primarily, PQ-induced cytotoxicity and reactive oxygen species (ROS) were inhibited by an inhibitor of NADPH oxidase (NOX), indicating the toxic effect of PQ redox cycling. Further, dibucaine and cyclosporin A which respectively inhibit mitochondrial apoptosis-induced channels (MAC) and mitochondrial permeability transition pores (mPTP) were used and found to prevent PQ-induced mitochondrial dysfunction, such as decreased mitochondrial membrane potential and increased MMP, mitochondrial ROS, and pro-apoptotic factor release. Knockdown of bax and/or bak blocked PQ-induced mitochondrial clusterization of Bax and/or Bak and cytotoxicity, demonstrating the significance of MAC which is composed of Bax and/or Bak. This clusterization coincided with the release of mitochondrial apoptotic factors before there was an increase in inner MMP, indicating that MAC may precede mPTP formation. Besides, NOX inhibitor but not dibucaine attenuated the earlier PQ-induced cytosolic ROS formation or Bax and/or Bak clusterization indicating PQ redox cycling may account for MAC formation. In this model, we have resolved for the first that PQ cytotoxicity through redox cycling may sequentially result in increased outer (MAC) and inner (mPTP) MMP and suggested MMP could be implicated as a therapeutic target in treating neurodegenerative diseases like PD.

The efficacy of antimicrobial drugs against Mycobacterium tuberculosis , an intracellular bacterial pathogen, is generally first established by testing compounds against bacteria in axenic culture. However, inside infected macrophages, bacteria encounter an environment which differs substantially from broth culture and are subject to important host-dependent pharmacokinetic phenomena which modulate drug activity. Here, we describe how pH-dependent partitioning drives asymmetric antimicrobial drug distribution in M. tuberculosis- infected macrophages. Specifically, weak bases with moderate activity against M. tuberculosis (fluoxetine, sertraline, and dibucaine) were shown to accumulate intracellularly due to differential permeability and relative abundance of their ionized and nonionized forms. Nonprotonatable analogs of the test compounds did not show this effect. Neutralization of acidic organelles directly with ammonium chloride or indirectly with bafilomycin A1 partially abrogated the growth restriction of these drugs. Using high-performance liquid chromatography, we quantified the degree of accumulation and reversibility upon acidic compartment neutralization in macrophages and observed that accumulation was greater in infected than in uninfected macrophages. We further demonstrate that the efficacy of a clinically used compound, clofazimine, is augmented by pH-based partitioning in a macrophage infection model. Because the parameters which govern this effect are well understood and are amenable to chemical modification, this knowledge may enable the rational development of more effective antibiotics against tuberculosis.

This study determined the effects of melatonin (MEL) and its receptors on monochromatic light-induced bursal B-lymphocyte proliferation in broiler chickens. In vivo, green light (GL) enhanced the proliferation of B lymphocytes in bursas by 16.49% to 30.83% and the expression of MEL receptor subtypes 1a ( Mel1a ), Mel1b , and Mel1c receptors in bursas by 6.91% to 366.98% than other light colors. However, pinealectomy reduced these parameters and eliminated the differences between GL and other light groups. In vitro, the MEL-induced bursal B-lymphocyte proliferation was most suppressed by prazosin ( P = 0.001, selective Mel1c antagonist), followed by luzindole ( P = 0.022, nonselective Mel1a/Mel1b antagonist), but not by 4-phenyl-2-propionamideotetralin ( P = 0.144, selective Mel1b antagonist). Similarly, dibutyryl-cyclic adenosine monophosphate (cAMP; analog of cAMP; P = 0.017) but not 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate ( P = 0.736; activator of exchange protein directly activated by cAMP) significantly inhibited bursal B-lymphocyte proliferation. These results suggest that MEL mediates GL-induced bursal B-lymphocyte proliferation through Mel1c and Mel1a receptors but not Mel1b receptors by activating the cAMP/protein kinase A pathway.

Calcium-dependent calpains are a family of cysteine proteases that have been demonstrated to play key roles in both platelet glycoprotein Ibα shedding and platelet activation and altered calpain activity is associated with thrombotic thrombocytopenic purpura. Calpain activators induce apoptosis in several types of nucleated cells. However, it is not clear whether calpain activators induce platelet apoptosis. Here we show that the calpain activator dibucaine induced several platelet apoptotic events including depolarization of the mitochondrial inner transmembrane potential, up-regulation of Bax and Bak, down-regulation of Bcl-2 and Bcl-X(L), caspase-3 activation and phosphatidylserine exposure. Platelet apoptosis elicited by dibucaine was not affected by the broad spectrum metalloproteinase inhibitor GM6001. Furthermore, dibucaine did not induce platelet activation as detected by P-selectin expression and PAC-1 binding. However, platelet aggregation induced by ristocetin or α-thrombin, platelet adhesion and spreading on von Willebrand factor were significantly inhibited in platelets treated with dibucaine. Taken together, these data indicate that dibucaine induces platelet apoptosis and platelet dysfunction.

Butyrylcholinesterase (BChE) metabolizes the paralytic succinylcholine. Extended paralysis occurs in people with inherited BChE variants that may be identified by measuring BChE activity with and without the inhibitor dibucaine to calculate a dibucaine number (DN). Accurate phenotyping requires phenotype-specific BChE and DN reference intervals. We investigated the concordance between the biochemical BChE phenotype and the BCHE genotype to establish interpretive criteria for biochemical results. DNA was extracted from 45 serum specimens for which BChE activity and DN had been determined. The BCHE gene coding region was amplified and sequenced. Phenotype-genotype concordance and discordance occurred in 16 (36%) and 15 (33%) of specimens, respectively. A phenotype could not be assigned for 14 specimens (31%). An incorrectly assigned phenotype did not change the risk of prolonged paralysis or implied a slightly increased risk when there was none. Accurate BChE phenotyping is difficult using only enzyme activity and DN. The combination of biochemistry and BCHE genotype could improve the assessment of patient risk.

A wide range of cationic amphiphilic drugs (CADs) from different therapeutic areas are known to cause phospholipidosis both in vivo and in vitro. Although the relevance of this storage disorder for human health remains uncertain, CADs have been repeatedly associated with clinical side effects, and as a result, phospholipidosis is of major concern for drug development in the pharmaceu- tical industry. An important unresolved question in this field is whether phospholipidosis is really linked to cellular toxicity. This work was focused on studying cellular responses associated with CAD-induced phospholipidosis in cultured mammalian kidney cells. Dibucaine (2-butoxy-N-(2-diethylaminoethyl)quinoline-4-carboxamide), an amide-type anesthetic with poorly defined cytotoxic effects, was used to induce phospholipidosis in Vero cells. The results from several assays that measure cell viability, proliferation, and morphological changes indicated that dibucaine-induced lysosomal phospholipidosis was accompanied by cellular defense responses such as transient growth arrest and autophagy, under mild stress conditions. Conversely, when tolerance limits were exceeded treated Vero cells underwent extensive and irreparable injury, leading ultimately to cell death. Our data provide additional information that may be of considerable interest for drug safety assessment.

SUMMARY Dibucaine, a local anesthetic, is known to induce flagellar excision in Chlamydomonas reinhardtii. Herein, we investigate whether other local anesthetics have similar effects. Tetracaine, bupivacaine, procaine, and lidocaine also caused flagellar excision, although their potencies were lower than that of dibucaine. Bupivacaine, procaine, and lidocaine induced a morphological change in flagella from a rod-like shape to a disk-like shape before flagellar excision. Except for lidocaine, these local anesthetics caused cell-wall shedding in addition to flagellar excision. The anesthetics in order of their median effective concentration (1-h EC50) for flagellar excision are as follows: dibucaine (1.37 × 10−5 M)

Rat sarcolemma preparations were incubated with some membranes stabilizers to study their effects on the (Na+,K+)- and Ca2+-ATPase activity. The drugs inhibited the enzymes with the same order of potency as in the earlier observed muscle contractures: chlorpromazine greater than dibucaine greater than propranolol greater than tetracaine greater than procaine (range 0.1-3.6 mM) (Roed & Brodal 1979). The contracture inducing effect of the stabilizers is suggested to be caused by a membrane depolarization due to the (Na+,K+)-ATPase inhibition. Ouabain inhibited the (Na+,K+)-ATPase activity in purified plasma membrane, but did not inherit the sarcolemma located ATPases or induce any contracture.

Dibucaine, fluoride and RO2-0683 inhibition studies were used to determine the serum cholinesterase (pseudocholinesterase) phenotypes at the E1 locus in a sample of 130 subject with Down's syndrome and fifty-three mentally retarded control subjects. No example of the Ef1 and Es1 allele was detected in either group, nor were any of the genotype E1a E1a detected. The gene frequency of E1a for the control group (0.0189) resembles closely that reported in the literature for normal European populations but in the Down's group was significantly lower (0.0038). The means and distribution of the dibucaine, fluoride and RO numbers in both groups were similar to those reported in normal subjects. The presence of the additional C5 cholinesterase type heterozygous for a variant cholinesterase on the E2 locus) was detected after starch gel electrophoresis, and the frequency was found to be raised in both groups. Several possible environmental factors (age, sex, maternal age, etc.) were investigated to account for this finding but with no success.

Cholinesterase deficiency was detected in a young girl following an episode of postanesthesia apnea. Subsequently, plasma and serum cholinesterase levels and dibucaine numbers were determined on blood samples from 56 members of her extended family. Including the proband, three individuals were identified with severe cholinesterase deficiency and 12 were found to have mild abnormalities. The occurrence of two genetic variants regulating cholinesterase production, the "silent" gene and the atypical enzyme, is postulated to account for the unusual pattern of inheritance in this family. Screening family members of confirmed cases is essential to prevent the potentially fatal consequences of this hereditary disorder.

Summary Estimates of the Michaelis constants for usual5 and ‘atypical’ serum cholinesterase with α-naphthyl acetate as substrate were obtained. Expressed as m-moles α-naphthyl acetate per litre the values were: ‘usual’ enzyme 0-76 + 0-056, ‘atypical’ enzyme 0-58 + 0-057. The inhibition of the activity of the two enzymes by dibucaine, RO 2-0683, and fluoride was also examined using α-naphthyl acetate as substrate. It was found that, in the appropriate concentration range, the ‘usual’ enzyme was inhibited to a much greater degree than the ‘atypical’ enzyme by dibucaine and RO 2-0683. With fluoride, however, the difference, though significant, was much less marked. The findings are compared with similar data obtained when choline esters were used as substrates for the enzymes, and are considered in terms of the hypothesis that a structural difference at the ‘anionic’ site is the cause of the difference in the properties of the two enzymes.

The tetrameric glycoprotein butyrylcholinesterase (BChE; EC 3.1.1.8) is one of two enzymes that hydrolyze choline esters. The controlling gene (BCHE) is comprised of four coding exons and is located on chromosome 3q26. Based on BChE activity measurements in the presence and absence of dibucaine, usual (designated U) and atypical (designated A) gene products have been distinguished. Homozygotes for the A gene product are at risk for prolonged apnea following exposure to the surgical anesthetics succinylcholine or mivacurium. In this report, we detail biochemical and molecular investigations of succinylcholine sensitivity in a prairie Hutterite kindred. Our results establish that BChE activities in the family members are impacted by two distinct BCHE mutations, namely, c.209A>G p. D70G and c.1615G>A p. A539T. However, homozygotes for the c.209A>G mutation (i.e., atypical or A) are the only individuals whose BChE activity could lead to adverse reactions to succinylcholine. Interestingly, haplotype analysis of the chromosomal region containing BCHE indicates that the c.209A>G mutation is carried on a unique haplotype, suggesting that it was likely introduced into the population only once. Conversely, the c.1615G>A mutation is carried on various haplotypes and was likely introduced into the population more than once.

Anesthetics have in common the capacity to partition indiscriminately into all biological membranes and act as general perturbants of the membrane structure and function. However, anesthetic mechanisms have not yet been clarified. Some measurement techniques are used to investigate the interaction between anesthetic and biological materials. Thus, in this research we examined the utility of the electrode selectively sensitive to local anesthetics as a measurement technique of the interaction of the bovine serum albumin (BSA) with local anesthetics. The electromotive forces (EMF) of the local anesthetic dibucaine and tetracaine cation-selective electrodes were measured relative to an Ag-AgCl reference electrode in both the absence and presence of BSA at pH 5.5. In the absence of BSA, both the dibucaine and tetracaine electrodes showed a linear response with a Nernstian slope over the concentration of 10−5 mol dm−3. Most of local aneshetic molecules must exist as the protonated cation at pH 5.5, judging from the pKa values. In the presence of BSA, the response curve was shifted toward the higher concentration of anesthetics, which means a decrease of the concentration of free anesthetic cation due to binding to BSA. The binding isotherms of anesthetics to BSA were calculated from a pair of response curves. The slope of the binding isotherm increased steeply above a certain concentration of local anesthetics, which means that the binding form of local anesthetic into BSA is multiple-step.

This case discusses a drug–gene interaction involving the inborn genetic variability of plasma cholinesterase causing prolonged neuromuscular weakness after succinylcholine.

This case discusses a drug–gene interaction between succinylcholine and a pseudocholinesterase variant, resulting in prolonged action of succinylcholine.

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman’s method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.

Many biochemical effects of local anesthetics are expressed in Ca2+-dependent processes [Volpi M., Sha'afi R. I., Epstein P. M., Andrenyak P. M., and Feinstein M. B. (1981) Proc. Natl. Acad. Sci. USA 78, 795–799]. In this communication we report that local anesthetics (dibucaine, tetracaine, lidocaine, and procaine and the analogue quinacrine) inhibit the Ca2+-dependent and the Mg2+-dependent ATPase activity of rat brain synaptosomes and of membrane vesicles derived from them by osmotic shock. This inhibition is induced by concentrations of these drugs close to their pharmacological doses, and a good correlation between K0.5 of inhibition and their relative anesthetic potency is found. The Ca2+-dependent ATPase is more selectively inhibited at lower drug concentrations. The physiological relevance of these findings is discussed briefly.

We report the case of a prolonged neuromuscular blockade in an 18-month-old age girl following administration of a usual dose of succinylcholine. The diagnosis was highly suggested by the clinical history while cholinesterase activity was included in adult normal values but below values of a personal series of 41 small children. The familial analysis of dibucaine and fluoride number confirmed the hypothesis of an atypical variant (AA phenotype). The cholinesterase activity is higher in small children than in adult and has to be analysed according to the age.

Although local anesthetics (LA) are considered primarily Na+-channel blockers in the past decade, an alternative action of LA as inhibitors of fusion among compartments of the endocytic/exocytic pathways was described. In epimastigote forms of Trypanosoma cruzi, we observed that 50 mM dibucaine reduced the rates of uptake of bovine serum albumin (BSA) and immunoglobulin to 60% of control values in addition to the delay of exocytosis of cysteine proteases. Fusion among endocytic compartments was not inhibited in the presence of dibucaine because previously labeled reservosomes was loaded with a second label in sequential pulse-chase experiments. However, dibucaine reduced the degradation of BSA-gold complex in the reservosomes, which was not caused either by an inhibition of the whole proteolytic activity of the parasite or by a reduction on the expression levels of cruzipain. The immunocytochemical analysis suggested that the inhibition of the degradation of gold-labeled BSA in reservosomes could be due to a subversion of the regular traffic of proteases toward the reservosomes in dibucaine-treated cells.

Blue light controls the sexual life cycle of Chlamydomonas, mediated by phototropin, a UV-A/blue-light receptor that plays a prominent role in multiple photoresponses. By using fractionation experiments and immunolocalization studies, this blue-light receptor, in addition to its known localization to the cell bodies, also was detected in flagella. Within the flagella, it was completely associated with the axonemes, in striking contrast to the situation in higher plants and the Chlamydomonas cell body where phototropin was observed in the plasma membrane. Its localization was not perturbed in mutants lacking several prominent structural components of the axoneme. This led to the conclusion that phototropin may be associated with the outer doublet microtubules. Analysis of a mutant (fla10) in which intraflagellar transport is compromised suggested that phototropin is a cargo for intraflagellar transport. The blue-light receptor thus seems to be an integral constituent of the flagella of this green alga, extending the list of organisms that harbor sensory molecules within this organelle to unicellular algae.

The effects of procaine and some relatedagents on the growth rate of thecyanobacterium Synechococcusleopoliensis were investigated underphotoautotropic growth conditions and thedata compared with those for selectedphytohormones. Low concentrations(0.1–1 μM) of synthetic localanaesthetics (procaine, procainamide,tetracaine, lidocaine, dibucaine) enhancedphotoautotrophic growth. Lidocaine was themost effective growth stimulant, followedin decreasing order by procainamide,tetracaine, dibucaine, and procaine.Increases in growth rate were comparable tothose elicited by similar concentrations ofthe auxins indole-3-acetic acid and2,4-dichlorophenoxyacetic acid and of thecytokinin kinetin. Quantitativestructure-activity relationships betweenthe anaesthetics and phytohormones weresought. Score plots of principal componentsformulated from twelve moleculardescriptors suggest that the proliferativeeffects evoked by the anaesthetics andphytohormones may result from similaraffinities for binding receptor sites onthe cyanobacterial cell surface.Cyanobacterial growth assays couldtherefore be utilised as screens forcompounds with potential applications inagriculture and medicine.evolution

SummaryPhosphatidylserine exposure in platelets is required for normal haemostasis and is also a hallmark of apoptosis. It results from activation of a phospholipid scramblase, which has been shown to be differently stimulated by Ca2+-influx and during apoptosis, thus suggesting that mitochondria may be involved in phosphatidylserine exposure in platelets. It is also well known that local anaesthetics can expose phosphatidylserine in platelets and affect the mitochondrial metabolism in other cells. Thus, the present study was undertaken to evaluate the specific involvement of mitochondria in phosphatidylserine exposure in platelets. For this purpose, we stimulated phosphatidylserine exposure by local anaesthetics and avoided any external Ca2+-influx by performing all experiments in the absence of added Ca2+. We report that phosphatidylserine exposure, induced by the lipophilic local anaesthetics dibucaine and tetracaine, was accompanied by depolarization of the mitochondrial membrane, cytochrome c release, calpain-processing of caspases 9 and 3 to active enzymes, as well as a prolonged increase in both cytosolic and mitochondrial Ca2+ concentrations. In contrast, in the absence of extracellular Ca2+, the Ca2+-ionophore A23187 induced a smaller transient increase in both cytosolic and mitochondrial Ca2+ concentrations, but did not induce any other phenomena, nor phosphatidylserine exposure. However, phosphatidylserine exposure and depolarization induced by dibucaine still occurred in spite of inhibition of intracellular Ca2+ elevation. Thus we conclude that phosphatidylserine exposure in platelets is associated with mitochondrial apoptotic-like events. Therefore, we propose that mitochondria engagement in an apoptotic pathway in platelets could lead to PS exposure without the participation of Ca2+.

Decreased activity of plasma cholinesterase is responsible for prolonged apnea during anesthesia using neuromuscular blockers such as suxamethonium and mivacurium. More than 20 mutations have been identified so far in the BCHE gene resulting in impaired plasma cholinesterase activity. Biochemical tests are not always able to differentiate between pathological and normal sera; hence in some cases unanticipated complications can still occur during anesthesia even after measurements of enzyme activity and dibucaine numbers within the normal range. Therefore, molecular genetic testing is required for the accurate diagnosis of this deficiency. Here we present a study of plasma cholinesterase activity and BCHE genotyping of patients with a history of prolonged neuromuscular block and most of their pedigrees. All four exons of the BCHE gene were directly sequenced from samples and a number of mutations responsible for the reduction of plasma cholinesterase activity were identified. In most cases the atypical mutation in exon 2 (nt 209A --> G, Asp70 --> Gly) was found together with the K-variant mutation in exon 4 (nt 1615G --> A, Ala539 --> Thr), which is in good agreement with previous data suggesting that these mutations along with two others (at nt -116 and nt 1914) are in linkage disequilibrium.

Local anaesthetics, in addition to anaesthesia, induce the synthesis of heat shock proteins (HSPs), sensitize cells to hyperthermia, and increase the aggregation of nuclear proteins during heat shock. Anaesthetics are membrane active agents, and anaesthesia appears to be due to altered ion channel activity; however, the direct effect of heat shock is protein denaturation. These observations suggest that local anaesthetics may sensitize cells to hyperthermia by interacting with and destabilizing membrane proteins such that protein denaturation is increased. It is shown, using differential scanning calorimetry (DSC), that the local anaesthetics procaine, lidocaine, tetracaine and dibucaine destabilize the transmembrane domains of the Ca2+ -ATPase of sarcoplasmic reticulum and the band III anion transporter of red blood cells. The transmembrane domain of the Ca2+ -ATPase has a transition temperature (Tm) of denaturation of 61 degrees C which is decreased, for example, to 53 degrees C by 15 mM lidocaine. The degree of destabilization (deltaTm) by each anaesthetic is proportional to the lipid to water partition coefficient, and the increased sensitization by anaesthetics with larger partition coefficients and at higher pH suggests that the uncharged forms of the anaesthetics are responsible for destabilization. A Hill analysis of deltaTm for the Ca2+ -ATPase as a function of the concentration of anaesthetic in water gives dissociation constants (Kd) on the order of 10(-4) M, if binding occurs directly from the aqueous phase. This demonstrates moderate affinity binding. However, dissociation constants of 1-3 M are obtained, if binding occurs through the lipid phase, which demonstrates low affinity binding. Thus, the interaction of local anaesthetics with the Ca2+ -ATPase may be moderately specific or non-specific depending on the mechanism of interaction. The observation that local anaesthetics also destabilize the transmembrane domain of the band III protein of erythrocytes suggests that destabilization of transmembrane proteins is a general property of anaesthetics, which is at least in part a mechanism of sensitization to hyperthermia.

Role of phospholipase A 2 in the cytotoxic effects of oxalate in cultured renal epithelial cells. Background Oxalate, a common constituent of kidney stones, is cytotoxic for renal epithelial cells. Although the exact mechanism of oxalate-induced cell death remains unclear, studies in various cell types, including renal epithelial cells, have implicated phospholipase A 2 (PLA 2 ) as a prominent mediator of cellular injury. Thus, these studies examined the role of PLA 2 in the cytotoxic effects of oxalate. Methods The release of [ 3 H]-arachidonic acid (AA) or [ 3 H]-oleic acid (OA) from prelabeled Madin-Darby canine kidney (MDCK) cells was measured as an index for PLA 2 activity. The cell viability was assessed by the exclusion of ethidium homodimer-1. Results Oxalate exposure (175 to 550 μM free) increased the release of [ 3 H]-AA in MDCK cells but had no effect on the release of [ 3 H]-OA. Oxalate-induced [ 3 H]-AA release was abolished by arachidonyl trifluoromethyl ketone (AACOCF 3 ), a selective inhibitor of cytosolic PLA 2 (cPLA 2 ), but was not affected by selective inhibitors of secretory PLA 2 and calcium-independent PLA 2 . The [ 3 H]-AA release could be demonstrated within 15 minutes after exposure to oxalate, which is considerably earlier than the observed changes in cell viability. Furthermore, AACOCF 3 significantly reduced oxalate toxicity in MDCK cells. Conclusions Oxalate increases AA release from MDCK cells by a process involving cPLA 2 . In addition, based on the evidence obtained using a selective inhibitor of this isoform, it would appear that the activity of this enzyme is responsible, at least in part, for the cytotoxic effects of oxalate. The finding that oxalate can trigger a known lipid-signaling pathway may provide new insight into the initial events in the pathogenesis of nephrolithiasis.

The time course of inhibition of butyrylcholinesterase (EC 3.1.1.8) by the dimethylcarbamate Ro 02-0683 in sera taken from patients heterozygous for the usual (U), atypical (A), K or J variant was followed using propionylthiocholine as substrate. Data obtained were used to determine rate constants of inhibition together with the contribution made by each variant to total enzyme activity. The findings substantiate earlier reports that J and K mutations lead to quantitative changes in the concentration of usual enzyme in contrast to the qualitative changes of the atypical variant. The contribution of the atypical enzyme to the total activity in serum from UA, AK and AJ heterozygotes was respectively 17-20, 24-31 and 34-53 %. The altered ratios of atypical to usual, K or J enzyme in UA, AK and AJ together with the constants on the usual enzyme alone explain the differences in observed inhibitor numbers which enable these heterozygotes to be identified in terms of enzyme kinetics.

Assays of radiosodium uptake into mouse brain vesicles and the binding of [ 3 H]batrachotoxinin A 20-α-benzoate (BTX-B) were used to compare the actions of six dihydropyrazole (3-aryl-1-arylcarbamoyl-2-pyrazoline) insecticides on mouse brain sodium channels. The relative potencies of the six dihydropyrazoles as inhibitors of either pumiliotoxin B-stimulated sodium uptake measured in the presence of scorpion ( Leiurus quinquestriatus ) venom or veratridine-stimulated sodium uptake were closely correlated with the relative potencies of these compounds as inhibitors of the binding of BTX-B to mouse brain sodium channels. A comparison of the enantiomers of the most potent dihydropyrazole, RH 3421, as inhibitors of radiosodium uptake showed that the (−) enantiomer of RH 3421 was approximately six-fold more potent than the (+) enantiomer. The potencies of these dihydropyrazoles in these assays and the stereoselectivity observed in the action of enantiomers of RH 3421 are in good agreement with available information on insecticidal activity in this group of compounds. Assays of the combined effects of RH 3421 and dibucaine as inhibitors of BTX-B binding revealed mutually competitive interactions between these compounds. This finding is consistent with the existence of a common site of action for dihydropyrazoles and local anesthetics on the sodium channel. The results of these studies provide further evidence for the toxicological relevance of the effects of dihydropyrazoles on sodium channels.

A random population was screened for abnormal dibucaine and fluoride numbers (DN & FN) to find some common mutations in butyrylcholinesterase ( BCHE) gene. Of 2375 unrelated individuals, 10 were found to have low DN and FN and were selected for further studies. DNA analysis of these hypocholinesterasemics revealed that seven patients were heterozygous for missense mutation at codon 330 (TTA to ATA; BCHE*330I). The frequency of BCHE*330I mutation was calculated to be at least 0.29% among the Japanese. On the other hand, two novel mutations were found in three families and two individuals including probands whose enzyme activity was very low (silent gene). Polymerase chain reaction and single stranded conformation polymorphism (PCR-SSCP) and restriction fragment length polymorphism (PCR-RFLP) were used for identification of the common and known mutation types such as BCHE*250P (ACT to CCT), BCHE*365R (GGA to CGA), and BCHE*539T (GCA to ACA; K-polymorphism), whereas PCR-SSCP was used in combination with direct DNA sequencing for new mutations like BCHE*446V (TTT to GTT) and BCHE*451X (GAA to TAA).

Chlorpromazine and prochlorperazine have previously been shown to enhance the susceptibility of Burkholderia cepacia to aminoglycosides. To screen other non-antibiotic drugs containing similar amine (-N-CH 3 ) groups, we examined a range of such agents that are in current clinical use for the treatment of non-infectious diseases, in combination with antibiotics that are ineffective against B. cepacia. At a concentration of 0.2 mM, theobromine, theophylline, trifluoperazine, fluophenazine and coumarin-152 significantly reduced (by fourfold) the MICs of gentamicin and ceftazidime. Theobromine and theophylline also reduced the MICs of amikacin and azithromycin.

We noticed a Japanese male showed low serum butyrylcholinesterase(BCHE) activity on health examination. The phenotyping analysis revealed a reduced dibucaine number (DN) and an especially low fluoride number (FN), similar to an FS phenotype. A homozygous missense mutation, a T to A transversion at nucleotide 988, was identified in his BCHE gene. This mutation resulted in the replacement of leucine by isoleucine at codon 330 (L330I). DN and FN of recombinant BCHE(L330I) secreted by human fetal kidney cells were compared to recombinant wild-type(usual gene)BCHE and normal serum BCHE. These results showed this amino acid substitution of BCHE, Leu330 to Ile, really caused the abnormal DN and FN. We conclude that the BCHE L330I mutation is a fluoride-resistant gene, a Japanese type fluoride-resistant gene.

1 Glutamate receptor activation has been previously shown to result in mitochondrial depolarization and activation of the mitochondrial permeability transition pore in cultured neurones. In this study, we characterized the effects of two putative permeability transition inhibitors, namely trifluoperazine and dibucaine, on mitochondrial depolarization in rat intact, cultured forebrain neurones. 2 Permeability transition was monitored by following mitochondrial depolarization in neurones loaded with the mitochondrial membrane potential-sensitive fluorescent indicator, JC-1. Trifluoperazine (10–20 μM) and dibucaine (50–100 μM) inhibited or delayed the onset of glutamate-induced permeability transition. 3 We also investigated the effects of trifluoperazine and dibucaine on neuronal recovery from glutamate-induced Ca2+ loads. Trifluoperazine affected Ca2+ recovery in a manner similar to the mitochondrial Na+/Ca2+ exchange inhibitor, CGP-37157, while dibucaine had no apparent effect on Ca2+ recovery. Therefore, inhibition of permeability transition does not appear to be involved in Ca2+ recovery from glutamate-induced Ca2+ loads. 4 Trifluoperazine and dibucaine did not inhibit [3H]-dizocilpine binding at the concentrations that prevented mitochondrial depolarization. 5 These studies suggest that trifluoperazine and dibucaine inhibit permeability transition in intact neurones. Trifluoperazine also appears to inhibit mitochondrial Na+/Ca2+ exchange. These drugs should prove to be valuable tools in the further study of the role of mitochondrial permeability transition in glutamate-induced neuronal death. British Journal of Pharmacology (1997) 122, 803–808; doi:10.1038/sj.bjp.0701442

Peroxide formation has been implicated in impairment of motor function by cyanide which occurs in both animals and man. The present study employs the neuronal model, rat pheochromocytoma (PC12) cells to evaluate peroxidation as a toxic mechanism of cyanide. Confocal imaging shows that peroxides form within a few seconds in cell cytoplasm after cyanide exposure and continue to accumulate over a period of several minutes. Peroxide generation by cyanide is decreased to about 50% by phospholipase A2 inhibitors indicating involvement of arachidonic acid in the oxidative process. Also antioxidant defense enzymes (CuZn superoxide dismutase and especially catalase) in PC12 cells are inhibited by cyanide. It appears that peroxide accumulation after cyanide treatment involves both inhibition of breakdown and increased production. Furthermore, both peroxide accumulation and cell death induced by cyanide in PC12 cells are blocked by an antioxidant (ascorbate). These data support the hypothesis that the cytotoxic action of cyanide is related in part to an oxidative process.

Dibucaine, a local anesthetic known to interact with cell membranes, induced apoptosis in SK-N-MC human neuroblastoma cells in a dose-dependent manner. Apoptosis was demonstrated by direct visualization of morphological nuclear changes using a DAPI staining technique and confirmed by the production of characteristic ladder patterns of DNA fragmentation on gel electrophoresis. At concentrations which induced apoptosis, dibucaine significantly altered membrane fluidity, indicating that fluidity may be a major target for the cytotoxic action of dibucaine. Also, dibucaine increased intracellular calcium levels more effectively in calcium-containing Krebs–Ringer buffer than in calcium-free Krebs–Ringer buffer. Removal of extracellular calcium or addition of antioxidants or protein synthesis inhibitor effectively blocked dibucaine-induced apoptosis. These results suggest that membrane damage, intracellular calcium levels, and oxygen free radicals may be involved in the apoptosis induced by dibucaine.

An unstable variant of human butyrylcholinesterase (BChE) is described in four apparently unrelated individuals sensitive to succinylcholine. Sequencing of genomic DNA revealed a single nucleotide substitution which results in the replacement of amino acid residue Gly115 by Asp. This variant can be recognized by its increased instability under extremes of temperature such as heating and also freezing and thawing, both in homozygous and heterozygous states. When in heterozygous combination with the Atypical variant, it produces dibucaine and fluoride numbers which are intermediary between those of Atypical homozygotes and heterozygotes. After repeated freezing and thawing, however, these values approach those of homozygous Atypical plasma. Measurement of activity and immunoreactive BChE protein in plasma of individuals representing different combinations of this allele indicated that the presence of the Usual or Atypical enzymes seems to partially protect this variant from denaturation in vivo. Phenotyping fresh serum or plasma samples, before they are frozen, is critical for the identification of this, and possibly some other, unstable variants.

Cells that express wild-type influenza hemagglutinin (HA) fully fuse to RBCs, while cells that express the HA-ectodomain anchored to membranes by glycosylphosphatidylinositol, rather than by a transmembrane domain, only hemifuse to RBCs. Amphipaths were inserted into inner and outer membrane leaflets to determine the contribution of each leaflet in the transition from hemifusion to fusion. When inserted into outer leaflets, amphipaths did not promote the transition, independent of whether the agent induces monolayers to bend outward (conferring positive spontaneous monolayer curvature) or inward (negative curvature). In contrast, when incorporated into inner leaflets, positive curvature agents led to full fusion. This suggests that fusion is completed when a lipidic fusion pore with net positive curvature is formed by the inner leaflets that compose a hemifusion diaphragm. Suboptimal fusion conditions were established for RBCs bound to cells expressing wild-type HA so that lipid but not aqueous dye spread was observed. While this is the same pattern of dye spread as in stable hemifusion, for this “stunted” fusion, lower concentrations of amphipaths in inner leaflets were required to promote transfer of aqueous dyes. Also, these amphipaths induced larger pores for stunted fusion than they generated within a stable hemifusion diaphragm. Therefore, spontaneous curvature of inner leaflets can affect formation and enlargement of fusion pores induced by HA. We propose that after the HA-ectodomain induces hemifusion, the transmembrane domain causes pore formation by conferring positive spontaneous curvature to leaflets of the hemifusion diaphragm.

A simple, scalable, and fast procedure for the isolation of Chlamydomonas flagella is described. Chlamydomonas can be synchronously deflagellated by treatment with chemicals, pH shock, or mechanical shear. The Basic Protocol describes the procedure for flagellar isolation using dibucaine to induce flagellar abscission; we also describe the pH shock method as an Alternate Protocol when flagellar regeneration is desirable. Sub-fractionation of the isolated flagella into axonemes and the membrane + matrix fraction is described in a Support Protocol.

We show with intact carbon-assimilating chloroplasts the induction of two types of rapidly reversible non-photochemical fluorescence quenching, distinguishable by the effects of ascorbate, de-epoxidation and dibucaine. In the presence of ascorbate, fluorescence quenching is antenna type and obligatory on violaxanthin de-epoxidation; in the absence of ascorbate, fluorescence quenching is reaction-center type and unrelated to violaxanthin de-epoxidation. The latter quenching type is inhibited by ascorbate, characterizing it as donor-side Photosystem II (PS II) inhibition. Consistent with current concepts of down-regulation of PS II efficiency by non-radiative energy dissipation, both types of quenching vary inversely to the putative ATP requirement of the carbon-substrate acceptors, although less clearly in the presence of ascorbate, presumably due to pseudocyclic electron transport by the Mehler-ascorbate-peroxidase reaction. It is concluded from the effects of dibucaine that reaction-center and antenna types of quenching are related to protons in different thylakoid compartments, the former to domains de-localized in the lumen and the latter to domains localized in the membrane. Antenna quenching, however, also requires lumen acidification for the obligatory violaxanthin de-epoxidase activity. No antenna quenching that is unrelated to de-epoxidation was observed. Since ascorbate is usually present in leaves, these results suggest that the prevailing mechanism for energy-dependent non-photochemical fluorescence quenching in vivo is de-epoxidation-dependent antenna quenching.

Effects of basic glycoside antibiotic aculeximycin (ACM) on the oxidative phosphorylation of rat-liver mitochondria were examined. ACM was shown to be a potent uncoupler of the oxidative phosphorylation. To cause the same extent of respiration release, higher concentration of ACM was required in phosphate (Pi)-free medium than in Pi medium. During the uncoupling caused by ACM in Pi medium, large amplitude swelling and oxidation of intramitochondrial NAD(P)H occurred, indicating that ACM remarkably enhances permeability of the inner mitochondrial membrane. The Pi uptake via Pi/H+ symporter was shown to play an important, but not essential, role in the uncoupling by ACM, indicating the increase in membrane permeability is mostly due to acceleration of Pi/H+ influx through Pi/H+ symporter activated by ACM. ACM is the first naturally occurring antibiotic, to our knowledge, which activates Pi/H+ symporter. However, since the inhibition of Pi/H+ symporter by N-ethylmaleimide did not completely abolish the uncoupling activity of ACM, and ACM induced the uncoupling even in Pi-free medium, an increase in the membrane permeability for other ions, such as Na+ and K+, due to a different action mechanism has also to be considered. On the other hand, positively charged amine local anesthetics, like dibucaine, prevented the uncoupling activity by ACM in both Pi and Pi-free medium. The uncoupling activity of N-diacetylated ACM lacking free amino groups was ca. 1/120th that of ACM, indicating that positively charged amino groups are important for the uncoupling activity. It is suggested that some specific interactions between positively charged amino groups of ACM and the binding site, which is probably negatively charged, are triggers that affect the permeability of the inner mitochondrial membrane. Amine local anesthetics may mask the negative charge of the binding site, thereby interfering with ACM binding.

The purpose of these experiments was to determine the reversibility of α-chaconine and α-solanine inhibition of human plasma butyrylcholinesterase (BuChE). For the substrate α-naphthylacetate, optimal assay conditions were 0.50 M sodium phosphate buffer and a substrate concentration of 3–5 × 10 −4 M . Dibucaine (1 × 10 −5 M ) indicated the usual phenotype for all subjects; α-chaconine and α-solanine at 2.88 × 10 −6 M inhibited BuChE about 70 and 50%, respectively. One- and 24-hr incubations at 1 × 10 −5 M with α-chaconine, α-solanine, paraoxon, eserine, and ethanol yielded reversible inhibition with dilution except for paraoxon. Twenty-four-hour dialyses of incubations showed no inhibition except for paraoxon. PAGE enzyme activity gels of 1- and 24-hr incubations also showed no inhibition except for paraoxon. α-Chaconine and α-solanine are reversible inhibitors of human butyrylcholinesterase. At estimated tissue levels, α-chaconine, α-solanine, and solanidine inhibited BuChE 10–86%. In assays which combined α-chaconine, α-solanine, and solanidine, inhibition of BuChE was less than additive. No inhibition of albumin α-naphthylacetate esterase (an arylesterase) was noted with any inhibitor. The importance of these data to adverse toxicological effects of potato alkaloids is discussed..

Background Mivacurium chloride is a bis-benzylisoquinolinium nondepolarizing neuromuscular blocking agent, hydrolyzed by butyrylcholinesterase (PCHE). The dose-response relationships for PCHE after mivacurium have not been studied. Therefore, this study was designed to establish dose-response relationships for PCHE as an antagonist of mivacurium-induced neuromuscular blockade. Methods Forty-eight physical status 1 adults were given 0.15 mg/kg mivacurium during fentanyl-thiopental-nitrous oxide-isoflurane anesthesia. Train-of-four (TOF) stimulation was applied to the ulnar nerve every 12 s, and the force of contraction of the adductor pollicis muscle was recorded. When spontaneous recovery of first twitch height (T1) reached 10% of its initial control value, exogenous PCHE equivalent to activity present in 2.5, 5, 7.5, 15, or 25 ml/kg of human plasma was administered by random allocation to 40 patients. Neuromuscular function in another eight subjects was allowed to recover spontaneously. Two blood samples were taken for determination of plasma cholinesterase activity. The first sample was taken before induction of anesthesia, and the second sample was taken when the TOF ratio had recovered to 0.75. Dibucaine and fluoride numbers were determined from the first assay. Results Administration of PCHE produced significant increases in PCHE activity in all patients. The larger the dose, the greater was the resultant plasma activity. Human PCHE produced a dose-dependent antagonism of mivacurium-induced neuromuscular blockade and the recovery times correlated inversely with PCHE activity (P < 0.01). The recovery of T1 was greater (P < 0.01) and time to attain a TOF ratio of 0.75 was shorter (P < 0.01) with any dose of PCHE than that observed in the spontaneous recovery group. After the administration of exogenous PCHE equivalent to activity present in 25 ml/kg of human plasma, recovery of TOF ratio to 0.75 or more was observed in all patients in less than 10 min and time to attain a TOF ratio of 0.75 was 55% shorter than the spontaneous recovery group (8.4 [7.1-9.7] vs. 18.7 [15.4-22] min; mean and 95% confidence intervals). Conclusions Administration of exogenous PCHE equivalent to activity present in 25 ml/kg of human plasma (in a 65-kg patient, this dose is equivalent to PCHE activity of 1,625 ml of adult human plasma) resulted in reliable antagonism of mivacurium-induced neuromuscular blockade. Nevertheless, because of the prohibitive cost of this compound, this reversal modality is unlikely to have a routine practical application at this time.

In 1973, a Cholinesterase Research Unit was established in Denmark (DCRU). The primary aim was to provide a central service for determining genotypes and activity of plasma cholinesterase (BChE) in patients showing abnormal response after succinylcholine. The purpose of the present study was, on the basis of 20 years experience with this Unit, to establish accurate reference intervals for BChE activity and inhibition values for the different genotypes of BChE. Also we wanted to evaluate the influence of age and sex on the BChE activity in genotypically normal patients. Plasma cholinesterase activity was measured using benzoylcholine as substrate. The genetic variations of the enzyme were identified using differential inhibitors, i.e.: Dibucaine, Sodium Fluoride, Succinylcholine, Urea and Ro-2-0683. We investigated 6,688 patients. The reference values for the 13 genotypes represented agree with previous findings. In genotypically normal patients, no age or sex differences were found in BChE activity in children below the age of 10 years. From the age of 10 years the activity decreased significantly in both males and females, the activity in females being significantly lower than in males. In females the activity was lowest in the age group 30-40 years, returning to prepuberty level at about 60 years of age. In males the activity decreased slightly up to 50-60 years of age. Hereafter the activity was stable or tended to increase slightly. Most genotypes could be recognized using the results of the different inhibition studies. We found the inhibitors Dibucaine, Sodium fluoride, Urea and Ro-2-0683 most helpful, whereas succinylcholine was of less value.(ABSTRACT TRUNCATED AT 250 WORDS)

The alteration of calcium content, Ca(2+)-ATPase activity, DNA content and DNA fragmentation in the nuclei of regenerating rat liver was investigated. Liver was surgically removed about 70% of that of sham-operated rats. The reduced liver weight by partial hepatectomy was completely restored at 3 days after the surgery. Regenerating liver significantly increased Ca(2+)-ATPase activity and DNA content in the nuclei between 1 and 5 days after hepatectomy. The nuclear calcium content was clearly increased from 2 days after hepatectomy. The increase of Ca(2+)-ATPase activity in regenerating liver was clearly inhibited by the presence of trifluoperazine (10 microM), staurosporine (2.5 microM) and dibucaine (10 microM), which are inhibitors of calmodulin and protein kinase, in the enzyme reaction mixture. However, the nuclear enzyme activity in normal rat liver was not significantly altered by these inhibitors. Meanwhile, the increase of nuclear DNA content in regenerating liver was completely blocked by the administration of trifluoperazine (2.5 mg/100 g body weight), suggesting an involvement of calmodulin. Now, the nuclear DNA fragmentation was significantly decreased in regenerating liver, suggesting that this decrease is partly contributed to the increase in nuclear DNA content. The present study clearly demonstrates that regenerating liver enhances nuclear Ca(2+)-ATPase activity and induces a corresponding elevation of nuclear calcium content. This Ca(2+)-signaling system may be involved in the regulation of nuclear DNA functions in regenerating rat liver.

Arachidonic acid release and the effect of phospholipase inhibitors on various types of cell injuries and death to rabbit renal proximal tubule suspensions were determined. Proximal tubules were exposed to the mitochondrial inhibitor antimycin A (0.1 microM), the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (1 microM FCCP), the oxidant tert-butyl hydroperoxide (0.5 mM TBHP), or the calcium ionophore ionomycin (5 microM) in the absence or presence of the putative phospholipase inhibitors dibucaine, mepacrine, chlorpromazine, or U-26384. The phospholipase inhibitors had no effect on the proximal tubule lactate dehydrogenase (LDH) release (a marker of cell death) produced by FCCP, antimycin A, or ionomycin after 1,2, or 2 hours of exposure, respectively. Only dibucaine and mepacrine decreased LDH release in TBHP-treated proximal tubules without decreasing TBHP-induced lipid peroxidation. Antimycin A and ionomycin did not release arachidonic acid from proximal tubules prelabeled with [1-14C] arachidonic acid. In contrast, TBHP released arachidonic acid from proximal tubules prior to the onset of cell death, and dibucaine and mepacrine decreased the TBHP-induced release. Thus, phospholipase inhibitors were cytoprotective in those injuries that produced arachidonic acid release. These results suggest that arachidonic acid release and phospholipase A2 activation play a contributing role in oxidant-induced renal proximal tubule cell injury and death but not in mitochondrial inhibitor- or calcium ionophore-induced proximal tubule cell injury and death.

In this study for the first time we used an electrophilic analog of tamoxifen, [ 3 H]tamoxifen aziridine. and demonstrated that it covalently and specifically binds to P-glycoprotein in multidrug resistant cells. Tamoxifen and its metabolites, N-desmethyltamoxifen and 4-hydroxytamoxifen, were potent inhibitors of [ 3 H]tamoxifen aziridine binding to P-glycoprotein with 4-hydroxytamoxifen > tamoxifen > N-desmethyltamoxifen. The multidrug resistance-related drugs inhibited [ 3 H]tamoxifen aziridine binding with vinblastine > vincristine > doxorubicin > actinomycin D, while colchicine enhanced the binding. Moreover, the multidrug resistance modulators verapamil, nicardipine, diltiazem, prenylamine, cyclosporin A, FK506, dibucaine, reserpine, monensin and progesterone were all potent inhibitors of [ 3 H]tamoxifen aziridine binding to P-glycoprotein. Our data provide the first evidence that [ 3 H]tamoxifen aziridine directly binds to P-glycoprotein and interacts with the binding sites for multidrug resistance-related drugs and modulators.

Hemocytes from the ascidian Ciona intestinalis exert in vitro Ca 2+ -dependent cytotoxic activity toward mammalian erythrocytes and K562 cells. To examine the lytic mechanism, hemocyte populations were separated (B1–B6 bands) through a Percoll discontinuous density gradient, the hemocyte cytotoxic activity (HCA) and the lytic activity of the hemocyte lysate supernatant (HLS) were assayed. In addition the separated hemocytes were cultured and the cell-free culture medium (CFM) assayed after 3 h culture. Results support that unilocular refractile hemocytes (URGs), enriched in B5, are cytotoxic. The B5-HLS contains lysins and the activity of B5-CFM shows that lysins can be released into a culture medium. The B5 activity was blocked by d -Galactose, α-Lactose, Lactulose, LacNAc, thiodigalactoside (TDG), l -Fucose, d -Mannose, d -Glucose, sphingomyelin (SM), and soluble phospholipase A2 (sPLA2) inhibitors (dibucain, quinacrine). Accordingly, HLS chemico-physical properties (alkaline medium, high thermostability, Ca 2+ -dependence, trypsin treatment, protease inhibitors) and SEM observations of the affected targets suggested that sPLA2 could be responsible for changes and large alterations of the target cell membrane. An apoptotic activity, as recorded by a caspase 3, 7 assay, was found by treating K562 cells with very diluted HLS. A lytic mechanism involving sPLA2 and lectins promptly released by URGs and morula cells respectively is suggested, whereas target cell membrane SM could be a modulator of the enzyme activity.

The factors controlling the relationship between the formation of the transthylakoid pH gradient, as measured by quenching of 9-aminoacridine fluorescence, and the induction of non-radiative de-excitation, measured by the non-photochemical quenching of chlorophyll fluorescence, have been measured in isolated spinach thylakoids. It was found that the following are potentiators of chlorophyll fluorescence quenching: high concentrations of Mg2+; pre-illumination to induce zeaxanthin synthesis; dibucaine. These treatments tended to be antagonistic to the action of antimycin, an inhibitor of quenching, in a way consistent with the LHCII model for fluorescence quenching. The reagent dibucaine allowed quenching to occur in the absence of a pH gradient as measured by the quenching of 9-aminoacridine fluorescence and markedly increased the rate of its formation. In all of these experiments quenching was correlated with the formation of an absorbance change with a peak near 530 nm. This absorbance band was shifted from 522 nm to 530 nm in the presence of zeaxanthin.

The traditional tests that have been used for the past 30 yr to determine plasma cholinesterase phenotype--measurement of esterase activity with a variety of substrates, dibucaine inhibition, fluoride inhibition, and Ro2-0683 inhibition--are inadequate for identifying some variants of this enzyme and leave many cases of prolonged response to succinylcholine unexplained. The application of the techniques of molecular genetics has permitted precise identification of plasma cholinesterase variants and has resulted in the discovery of previously unrecognized variants. It is now possible, in cases of prolonged response to succinylcholine resulting from genetically determined alterations in plasma cholinesterase, to ascertain the nature of the mutations in the alleles, and from them to deduce the structural changes in the enzymes responsible for the impairment in drug metabolism.