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Background: The mechanisms underlying gastrointestinal (GI) dysmotility with ulcerative colitis (UC) have not been fully elucidated. The enteric nervous system (ENS) plays an essential role in the GI motility. As a vital neurotransmitter in the ENS, the gas neurotransmitter nitric oxide (NO) may impact the colonic motility. In this study, dextran sulfate sodium (DSS)-induced UC rat model was used for investigating the effects of NO by examining the effects of rate-limiting enzyme nitric oxide synthase (NOS) changes on the colonic motility as well as the role of the ENS in the colonic motility during UC., Aim: To reveal the relationship between the effects of NOS expression changes in NOS-containing nitrergic neurons and the colonic motility in a rat UC model., Methods: Male rats ( n = 8/each group) were randomly divided into a control (CG), a UC group (EG1), a UC + thrombin derived polypeptide 508 trifluoroacetic acid (TP508TFA; an NOS agonist) group (EG2), and a UC + NG-monomethyl-L-arginine monoacetate (L-NMMA; an NOS inhibitor) group (EG3). UC was induced by administering 5.5% DSS in drinking water without any other treatment (EG1), while the EG2 and EG3 were gavaged with TP508 TFA and L-NMMA, respectively. The disease activity index (DAI) and histological assessment were recorded for each group, whereas the changes in the proportion of colonic nitrergic neurons were counted using immunofluorescence histochemical staining, Western blot, and enzyme linked immunosorbent assay, respectively. In addition, the contractile tension changes in the circular and longitudinal muscles of the rat colon were investigated in vitro using an organ bath system., Results: The proportion of NOS-positive neurons within the colonic myenteric plexus (MP), the relative expression of NOS, and the NOS concentration in serum and colonic tissues were significantly elevated in EG1, EG2, and EG3 compared with CG rats. In UC rats, stimulation with agonists and inhibitors led to variable degrees of increase or decrease for each indicator in the EG2 and EG3. When the rats in EGs developed UC, the mean contraction tension of the colonic smooth muscle detected in vitro was higher in the EG1, EG2, and EG3 than in the CG group. Compared with the EG1, the contraction amplitude and mean contraction tension of the circular and longitudinal muscles of the colon in the EG2 and EG3 were enhanced and attenuated, respectively. Thus, during UC, regulation of the expression of NOS within the MP improved the intestinal motility, thereby favoring the recovery of intestinal functions., Conclusion: In UC rats, an increased number of nitrergic neurons in the colonic MP leads to the attenuation of colonic motor function. To intervene NOS activity might modulate the function of nitrergic neurons in the colonic MP and prevent colonic motor dysfunction. These results might provide clues for a novel approach to alleviate diarrhea symptoms of UC patients., Competing Interests: Conflict-of-interest statement: All authors report no relevant conflicts of interest for this article., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

The enzyme responsible for the enantioselective production of (S)-1,1,1-trifluoro-2-propanol ((S)-TFP) from 1,1,1-trifluoroacetone (TFA) has been identified in Ogataea polymorpha NBRC 0799. We purified two carbonyl reductases, OpCRD-A and OpCRD-B from this strain, and revealed their characteristics. Both enzymes were specific to NADH, but the following characteristics were different: The molecular mass of subunit OpCRD-A was 40 kDa and that of OpCRD-B was 43 kDa. Amino acid sequences of both enzymes were only 21% identical. OpCRD-B contained 4 mol of zinc per mole of enzyme, but OpCRD-A did not. The optimal pH, temperature, pH stability, thermostability, and inhibitor specificity were also remarkably different. With regard to substrate specificity, both enzymes exhibited high reductase activity toward a wide variety of ketones, aldehydes and fluoroketones, and dehydrogenase activity toward 2-propanol and 2-butanol. The reductase activity was much higher than the dehydrogenase activity at acidic pH. OpCRD-A enantioselectively produced (S)-TFP from TFA, but OpCRD-B preferentially produced (R)-TFP. Thus, we concluded that OpCRD-A plays the main role in the production of (S)-TFP by a reaction of O. polymorpha NBRC 0799 cells and that OpCRD-A has great potential for efficient production of (S)-TFP, as it is an S-specific enzyme and does not catalyze the dehydrogenation of (S)-TFP.

Queensland fruit fly, Bactrocera tryoni (Q-fly), is a major pest of horticultural crops in eastern Australia. Lures that attract male Q-fly are important for detection of incursions and outbreaks, monitoring of populations, and control by mass trapping and male annihilation. Cuelure, an analog of naturally occurring raspberry ketone, is the standard Q-fly lure, but it has limited efficacy compared with lures that are available for some other fruit flies such as methyl eugenol for B. dorsalis. Melolure is a more recently developed raspberry ketone analog that has shown better attraction than cuelure in some field studies but not in others. A novel fluorinated analog of raspberry ketone, raspberry ketone trifluoroacetate (RKTA), has been developed as a potential improvement on cuelure and melolure. RKTA placed on laboratory cages containing 2-week-old Q-flies elicited strong behavioral responses from males. Quantification of Q-fly responses in these cages, using digital images to estimate numbers of flies aggregated near different lures, showed RKTA attracted and arrested significantly more flies than did cuelure or melolure. RKTA shows good potential as a new lure for improved surveillance and control of Q-fly.

Grass cell wall properties influence food, feed, and biofuel feedstock usage efficiency. The glucuronoarabinoxylan of grass cell walls is esterified with the phenylpropanoid-derived hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA). Feruloyl esters undergo oxidative coupling with neighboring phenylpropanoids on glucuronoarabinoxylan and lignin. Examination of rice (Oryza sativa) mutants in a grass-expanded and -diverged clade of BAHD acyl-coenzyme A-utilizing transferases identified four mutants with altered cell wall FA or p-CA contents. Here, we report on the effects of overexpressing one of these genes, OsAt10 (LOC_Os06g39390), in rice. An activation-tagged line, OsAT10-D1, shows a 60% reduction in matrix polysaccharide-bound FA and an approximately 300% increase in p-CA in young leaf tissue but no discernible phenotypic alterations in vegetative development, lignin content, or lignin composition. Two additional independent OsAt10 overexpression lines show similar changes in FA and p-CA content. Cell wall fractionation and liquid chromatography-mass spectrometry experiments isolate the cell wall alterations in the mutant to ester conjugates of a five-carbon sugar with p-CA and FA. These results suggest that OsAT10 is a p-coumaroyl coenzyme A transferase involved in glucuronoarabinoxylan modification. Biomass from OsAT10-D1 exhibits a 20% to 40% increase in saccharification yield depending on the assay. Thus, OsAt10 is an attractive target for improving grass cell wall quality for fuel and animal feed.

Rationale: It is recommended that harmful Biosafety Level 3 (BSL-3) bacteria be inactivated prior to identification by mass spectrometry, yet optimal effects of inactivation protocol have not been defined., Methods: Here, we compare trifluoroacetic acid inactivation (protocol A) with ethanol inactivation (protocol B) of Yersinia organisms prior to identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)., Results: The total number of peaks detected was 10.5 ± 1.7 for protocol A and 15.7 ± 4.2 for protocol B (ρ <0.001, ANOVA test). The signal-to-noise ratio for the m/z 6049 peak present in all of the tested Yersinia isolates was 9.7 ± 3.1 for protocol A and 18.1 ± 4.6 for protocol B (ρ < 0.001). Compared with spectra in our local database containing 48 Yersinia spp., including 20 strains of Y. pestis, the identification score was 1.79 ± 0.2 for protocol A and 1.97 ± 0.19 for protocol B (ρ = 0.0024)., Conclusions: Our observations indicate that for the identification of Yersinia organisms, ethanol inactivation yielded MALDI-TOF-MS spectra of significantly higher quality than spectra derived from trifluoroacetic acid inactivation. Combined with previously published data, our results permit the updating of protocols for inactivating BSL-3 bacteria., (Copyright © 2012 John Wiley & Sons, Ltd.)

The biodegradation of five pharmaceutical ingredients (PIs) of different therapeutic classes, namely antibiotics (trimethoprim, sulfametoxazole and ciprofloxacin), anti-inflammatory (diclofenac) and anti-epileptic (carbamazepine), by two distinct microbial consortia, was investigated. For the monitoring of biodegradation assays, a simple HPLC-DAD (High Performance Liquid Chromatography - Diode Array Detector) method was developed and validated. The separation of the target pharmaceuticals was performed using an environmental friendly mobile phase in a gradient mode of 0.1% triethylamine (TEA) in water acidified at pH 2.23 with trifluoroacetic acid (TFAA) and ethanol as organic solvent. The method revealed to be selective, linear and precise in the range of 1.0 to 30.0 μg/mL for all PIs. Biodegradation assays were performed using activated sludge and a bacterial consortium (able to degrade fluoroaromatic compounds) supplemented with the target PIs at a final concentration of 25 μg/mL. The results revealed that activated sludge removed the target compounds more efficiently than the bacterial consortium.

This article presents an improved method to detect D-glucosamine hydrochloride in health foods. A simple precolumn derivatization procedure with 7-flouro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) reagent was employed. The separation of the derivatized D-glucosamine hydrochloride (NBD-D-glucosamine hydrochloride) was performed using a mobile phase consisting of acetonitrile, potassium dihydrogen phosphate (0.01 mol/L), and trifluoroacetic acid (350:649.74:0.26, volume ratio) at a flow rate of 1.0 mL/min with the column temperature 35 °C. Under the optimum chromatographic conditions, the peak area of NBD-D-glucosamine hydrochloride compared with its absolute value of the peak area of NBD-D-glucosamine hydrochloride in a standard solution concentration range from 1.0 to 500.0 mg/L showed a good linear calibration (R = 0.9999). Recoveries, at spiked concentrations of 10.0, 40.0, and 500.0 mg/L, varied between 97.2% and 102.6% with relative standard deviations ranging from 0.4% to 1.5%. The present method provides sufficient sensitivity as reflected by the values of limit of detection (LOD) and limit of quantification (LOQ). LOD was determined from the signal-to-noise ratios (S/N) of NBD-D-glucosamine hydrochloride peak of at least 3 in the recovery test at 0.02 mg/L, and the estimated LOQ was 0.06 mg/L (S/N = 10). The proposed method was successfully applicable to detect D-glucosamine hydrochloride in health foods and drugs containing a variety of complex materials., (© 2011 Institute of Food Technologists®)

We report the first systematic synthesis of monodisperse rare-earth (RE=La to Lu, Y) fluoride and oxyfluoride nanocrystals with diverse shapes (trigonal REF3 triangular, truncated-triangular, hexagonal, and polygonal nanoplates; orthorhombic REF3 quadrilateral and zigzag-shaped nanoplates; cubic REOF nanopolyhedra and nanorods) from single-source precursors (SSP) of [RE(CF(3)COO)(3)] through controlled fluorination in oleic acid (OA)/oleylamine (OM)/1-octadecene (ODE). To selectively obtain REF3 or REOF nanocrystals, the fluorination of the RE-O bond to the RE-F bond at the nucleation stage was controlled by finely tuning the ratio of OA/ODE or OA/OM, and the reaction temperature. For phase-pure REF3 or REOF naocrystals, their shape-selective syntheses could be realized by further modifying the reaction conditions. The two-dimensional growth of the REF3 nanoplates and the one-dimensional growth of the REOF nanorods were likely due to the selective adsorption of the capping ligands on specific crystal planes of the nanocrystals. Those well-shaped nanocrystals with diverse geometric symmetries (such as D(3h), D(6h), C(2h), O(h), and D(nh)) displayed a remarkable capability to form self-assembled superlattices. By manipulating the solvent-substrate combination, the plate-shaped REF3 nanocrystals could form highly ordered nanoarrays by means of either the face-to-face formation or the edge-to-edge formation. By using this SSP strategy, we also obtained high-quality LaF3:Eu and LaF3:Eu/LaF3 triangular nanoplates that showed photoluminescent red emissions of Eu3+ ions sensitive to the surface effect.

Drug-induced liver injury (DILI) is a major safety concern in drug development. Its prediction and prevention have been hindered by limited knowledge of the underlying mechanisms, in part the result of a lack of animal models. We developed a mouse model of halothane-induced liver injury and characterized the mechanisms accounting for tissue damage. Female and male Balb/c, DBA/1, and C57BL/6J mice were injected intraperitoneally with halothane. Serum levels of alanine aminotransferase and histology were evaluated to determine liver injury. Balb/c mice were found to be the most susceptible strain, followed by DBA/1, with no significant hepatotoxicity observed in C57BL/6J mice. Female Balb/c and DBA/1 mice developed more severe liver damage compared with their male counterparts. Bioactivation of halothane occurred similarly in all three strains based on detection of liver proteins adducted by the reactive metabolite. Mechanistic investigations revealed that hepatic message levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta); IL-6, and IL-8 were significantly higher in halothane-treated Balb/c mice compared to DBA/1 and C57BL/6J mice. Moreover, a higher number of neutrophils were recruited into the liver of Balb/c mice upon halothane treatment compared with DBA/1, with no obvious neutrophil infiltration detected in C57BL/6J mice. Neutrophil depletion experiments demonstrated a crucial role for these cells in the development of halothane-induced liver injury. The halothane-initiated hepatotoxicity and innate immune response-mediated escalation of tissue damage are consistent with events that occur in many cases of DILI. In conclusion, our model provides a platform for elucidating strain-based and gender-based susceptibility factors in DILI development.

Identification of peptides derived from pancreatic islet and presented by type 1 diabetes-susceptible MHC class II molecules has great significance to elucidate the pathogenesis of type 1 diabetes. A bulk culture of Epstein-Barr virus-transformed B-cells, which were established from a 22-year-old type 1 diabetic woman with HLA-DR4 and -DQw8, was pulsed with the homogenate of a human embryonic pancreas-derived cell line 1B2C6, and another culture was not pulsed with antigen. Peptide fractions were obtained by treatment of affinity-purified HLA-DR and -DQ molecules with 0.1% trifluoroacetic acid, and were subjected to reverse-phase high performance liquid chromatography (RP-HPLC). The RP-HPLC profiles of peptides derived from DR molecules revealed three peaks that specifically appeared after pulsing, but no such peaks were obtained from DQ molecules. From one of these three peaks, a peptide that consisted of 14 amino acids (AKSXNHTXXNQXRK, where X represents the undetermined amino acids) was identified. This peptide was derived from heparin/heparan sulfate-interacting protein (HIP). Immunostaining of pancreatic sections using antiserum for HIP peptide revealed exclusive staining of the islets. Thus, HIP was identified as an islet protein naturally processed and presented by HLA-DR4 molecules.

Histone deacetylases (HDACs) are important enzymes for the transcriptional regulation of gene expression in eukaryotic cells. Deacetylation of epsilon-acetyl-lysine residues within the N-terminal tail of core histones mediates changes in both histone-DNA and histone-non-histone protein interactions. However, surprisingly little is known about the substrate specificities of different HDACs. Here, we use the epsilon-acyl moieties of epsilon-modified l-lysine in peptidic substrates as a probe to examine the active site cavity of HDACs and HDAC-like enzymes. Measurements were based on a fluorogenic assay with small synthetic substrates. Four different enzyme preparations were used derived from rat, human, and bacterial sources. None of the enzymes was able to utilize substrates with epsilon-acyl moieties larger than acetyl, except rat liver HDAC, which was the only enzyme to convert a substrate containing epsilon-propionyl-l-lysine. All enzymes exhibited a distinct enantioselectivity toward l-lysine-containing substrates except FB188 HDAH which also deacetylated Boc-d-Lys(epsilon-acetyl)-MCA. Moreover, all enzymes also exhibited a distinct specificity for the length of the lysine side chain; acetylated ornithine, which comprises one CH(2) unit less in the side chain, was not a substrate. In line with these results, only acetylcadaverin the metabolic degradation product of lysine but neither acetylputrescine (degradation product of ornithine) nor acetylspermidine strongly inhibited enzyme activity. Boc-l-Lys(epsilon-trifluoroacetyl)-MCA was observed to be a superior substrate for FB188 HDAH, Pseudomonas aeruginosa HDAH (PA3774), and particularly HDAC 8 compared to rat liver HDAC, and is the first suitable (synthetic) substrate for (human-derived) HDAC 8 reported to date. Altogether, the results reveal clear differences in substrate specificity between different HDACs as analyzed in the fluorogenic HDAC assay. Finally, we present the first candidates for HDAC-type-selective substrates that may be useful as biochemical tools to establish the function of particular pathways and to elucidate the role of distinct HDAC subtypes in cellular differentiation and cancer.

The interaction between different HO(z.rad;) radical scavengers in a three-component antioxidant system has been investigated by means of 19F-NMR spectroscopy. This system is composed of bovine serum albumin (BSA), trolox, and N-(4-hydroxyphenyl)-trifluoroacetamide (CF(3)PAF). The antioxidant capacity of BSA and trolox has been assessed by measuring the amount of trifluoroacetamide (TFAM) arising from the radical mediated decomposition of CF(3)PAF. When assayed separately, both trolox and BSA behaved as antioxidants, as they were effective to protect CF(3)PAF from HO* radical-mediated decomposition. By contrast, trolox enhanced the production of TFAM in the presence of BSA, thus behaving as a pro-oxidant. Urate, carnosine, glucose, and propylgallate showed antioxidant properties both with or without BSA. CF(3)PAF and trolox were found to bind to BSA with association constants in the order of 5 x 10(3)M(-1) and to compete for the same binding sites. These results have been discussed in terms of BSA-catalysed cross-reactions between trolox-derived secondary radicals and CF(3)PAF.

Background: Cytochrome P4502E1(CYP2E1)-mediated oxidation of halothane to a reactive intermediate (trifluoroacyl chloride) that covalently binds to hepatic proteins forming trifluoroacetylated neoantigens is believed to be the initiating event in a complex immunologic cascade culminating in antibody formation and severe hepatic necrosis ('halothane hepatitis') in susceptible patients. Trifluoroacyl chloride may also hydrolyze to the stable metabolite trifluoroacetic acid (TFA). CYP2E1 inactivation by disulfiram or its primary metabolite, diethyldithiocarbamate, inhibits human halothane oxidation to TFA in vitro and in vivo. Nevertheless, disulfiram effects on hepatic protein trifluoroacetylation by halothane in vivo are unknown. This investigation tested the hypotheses that disulfiram prevents halothane-dependent protein trifluoroacetylation in vivo, and that TFA represents a biomarker for hepatic protein trifluoroacetylation., Methods: Rats were pretreated with isoniazid (CYP2E1 induction), isoniazid followed by disulfiram (CYP2E1 inhibition), or nothing (controls), then anesthetized with halothane or nothing (controls). Plasma and urine TFA were quantified by ion HPLC; hepatic microsomal TFA-proteins were analyzed by Western blot., Results: CYP2E1 induction increased both TFA and TFA-protein formation compared with uninduced halothane-treated rats. Disulfiram, even after CYP2E1 induction, nearly abolished both TFA and TFA-protein formation. Pretreatments similarly affected both TFA and TFA-protein formation across all groups., Conclusions: Disulfiram inhibition of CYP2E1-mediated halothane oxidation prevents hepatic protein trifluoroacetylation. Based on the concordance between TFA and TFA-protein formation, TFA appears to be a valid biomarker for TFA-protein formation. Disulfiram inhibition of human halothane oxidation in vivo, previously assessed by diminished TFA formation, probably also confers inhibition of hepatic TFA-protein formation.

Peroxisome proliferators are a class of nongenotoxic rodent hepatocarcinogens that cause peroxisome proliferation and liver tumors when administered to rats and mice; but other species, including guinea pigs, dogs, and primates are less sensitive or refractory to the induction of peroxisome proliferation. Therefore, rodent peroxisome proliferators are not believed to pose a hepatocarcinogenic hazard to humans. Some peroxisome proliferators produce developmental toxicity in rats that is expressed as suppressed postnatal growth. To evaluate the relevance of the rat developmental effect to primates, groups of 4 lactating female Rhesus monkeys and their infants were exposed for 6 h/day, 7 days/week for 3 weeks to air or 1000 ppm HCFC-123. Animals were evaluated for clinical signs, body weights, clinical pathology parameters, and biochemical and pathological evaluations of liver biopsy samples. The effect of HCFC-123 exposure on milk quality (protein and fat concentration) was evaluated. The concentrations of HCFC-123 and the major metabolite, trifluoroacetic acid (TFA), were measured in the blood of the mothers and infants and in the milk. Exposure of monkeys to 1000 ppm HCFC-123 did not result in exposure-related clinical observations, or changes in body weight, appetence and behavior. There were no exposure-related effects on serum triglycerides, cholesterol, or glucose levels. HCFC-123 and TFA were present in milk, although maternal HCFC-123 exposure did not affect milk protein and fat content. In general, HCFC-123 was not detected in maternal or infant blood. TFA was detected in the majority of the mothers and TFA levels in infants ranged from 2 to 6 times higher than levels in the corresponding maternal blood. A pharmacokinetic analysis in a maternal animal indicated a peak concentration of TFA at approximately 1 h post-exposure, with a half-life of approximately 20 h. Liver microsomal P450 and peroxisome oxidase activities showed exposure-related decreases in CYP4A1 and CYP2E1 and acyl-CoA oxidase for animals exposed to HCFC-123. Microscopic evaluation of maternal liver from HCFC-123 exposed animals revealed mild to moderate centrilobular hepatocyte vacuolation, trace to mild centrilobular necrosis, and trace to mild subacute inflammation. The histopathological damage and altered hepatic biochemical activities produced by HCFC-123 in monkeys are not consistent with the HCFC-123 peroxisome proliferation response observed in rat livers. These findings demonstrate that HCFC-123 is not a peroxisome proliferator in adult Rhesus monkeys and postnatal exposure to HCFC-123 does not affect body weight of nursing infant monkeys.

Nitric oxide (NO) binds to the myoglobin (Mb) cavity mutant, H93G, forming either a 5- or 6-coordinate Fe--NO heme complex. The H93G mutation replaces the proximal histidine of Mb with glycine, allowing exogenous ligands to occupy the proximal binding site. In the absence of the covalently attached proximal ligand, NO could bind to H93G from the proximal side of the heme rather than the typical diatomic binding pocket on the distal side when the 5-coordinate complex forms. The question of whether NO binds on the distal or proximal side was addressed by (19)F NMR. Site-directed mutagenesis was used to introduce unique cysteine residues at the protein surface on either the distal (S58C) or proximal (L149C) side, approximately equidistant from and perpendicular to the heme plane of both wild-type and H93G Mb. The cysteine thiols were alkylated with 3-bromo-1,1,1-trifluoroacetone to attach a trifluoroacetyl group at the mutation site. (19)F NMR spectra of 5-coordinate, NO bound S58C/H93G and L149C/H93G double mutants depict peaks with line widths of 100 and 23 Hz, respectively. As fluorine peaks broaden with increasing proximity to paramagnetic centers, such as 5-coordinate Fe--NO, the (19)F NMR data are consistent with NO binding in the distal heme pocket of H93G, even in the absence of a sixth axial ligand. Additionally, (19)F NMR spectra are reported for deoxy, oxy, CO, met CN, and met H(2)O forms of the labeled cysteine mutants. These results demonstrate that the fluorine probes are sensitive to subtle conformational changes in the protein structure due to ligation and oxidation state changes of the heme iron in Mb.

This unit describes a number of methods for modifying cysteine residues of proteins and peptides by reduction and alkylation procedures. A general procedure for alkylation of cysteine residues in a protein of known size and composition with haloacyl reagents or N-ethylmaleimide (NEM) is presented, and alternate protocols describe similar procedures for use when the size and composition are not known and when only very small amounts of protein are available. Alkylations that introduce amino groups using bromopropylamine and N-(iodoethyl)-trifluoroacetamide are also presented. Two procedures that are often used for subsequent sequence analysis of the protein, alkylation with 4-vinylpyridine and acrylamide, are described, and a specialized procedure for 4-vinylpyridine alkylation of protein that has been adsorbed onto a sequencing membrane is also presented. Reversible modification of cysteine residues by way of sulfitolysis is described, and a protocol for oxidation with performic acid for amino acid compositional analysis is also provided. Gentle oxidation of cysteine residues to disulfides by exposure to air is detailed. Support protocols are included for recrystallization of iodoacetic acid, colorimetric detection of free sulfhydryls, and desalting of modified samples.

Two enzymatic methods commonly used in N-terminal sequence analysis of blocked proteins are presented in this unit; one uses pyroglutamate aminopeptidase for N(alpha)-pyrrolidone carboxyl-proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl-peptide hydrolase for N(alpha)-acyl-proteins blocked with other acyl groups. A Support Protocol describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl-peptide hydrolase must include fragmentation of the protein before unblocking can be carried out, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N-terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis. Protocols are also provided for unblocking N-terminally blocked proteins using acid-catalyzed hydrolysis or methanolysis, hydrazinolysis, and beta-elimination after acid-catalyzed N-O shift. Alternate protocols describe chemical removal of acetyl and longer-chain alkanoyl groups, as well as formyl groups to open the cyclic imide of pyrrolidone carboxylate.

The environmental fate of trichloro-, dichloro-, and monochloroacetic acids, and trifluoroacetic acid was investigated using field aquatic microcosms and laboratory sediment-water systems. Trifluoroacetic acid was extremely persistent and showed no degradation during a one-year field study, though it appeared to undergo transient partitioning within an unknown pond phase as the temperature of the surroundings was reduced. Of the three chloroacetic acids, trichloro had the longest residence time (induction and decay) (approximately 40 d), dichloro the shortest (approximately 4 d), and monochloro an intermediate residence time (approximately 14 d). Laboratory studies suggest that the biodegradation of trichloro-, dichloro-, and monochloroacetic acids leads primarily to the formation of chloride and oxalic, glyoxalic, and glycolic acids, respectively.

19F nuclear magnetic resonance (NMR) imaging and 19F NMR chemical-shift imaging (19F CSI) have been used to localize fluorinated compounds administered to stems of Ancistrocladus heyneanus and A. abbreviatus for the elucidation of biosynthetic pathways in living plants. This first application of 19F CSI on plants proved CSI to be a valuable technique for mapping fluorinated molecules in plants. Exemplarily using trifluoroacetate as a model compound allowed to select appropriate feeding methods and to optimize both concentration and duration of the application to the plant. The time course of the uptake and distribution of trifluoroacetate was monitored by both 19F imaging and 19F CSI. Fluorinated metabolites formed by uptake of 3-fluoro-3-deoxy-D-glucose were detected with 19F CSI.

An evaluation of derivatization procedures for the detection of beta(2)-agonists is presented. The study was performed with the beta(2)-agonists bambuterol, clenbuterol, fenoterol, formoterol, salbutamol, salmeterol and terbutaline. Different derivatizating agents were employed, aiming to obtain derivatives with high selectivity to be used in the gas chromatographic/mass spectrometric analysis of beta(2)-agonists in biological samples. Trimethylsilylation was compared with different agents and the role of some catalysts was evaluated. Acylation, combined trimethylsilylation and acylation, and the formation of cyclic methylboronates were also studied. Sterical hindrance caused by different substituents at the nitrogen atom of the beta-ethanolamine lateral chain of beta(2)-agonist molecules is mainly responsible for differences in the abundances of the derivatives obtained. The use of catalysts produces an increase in the derivatization yield, especially for compounds with low steric hindrance (substituents with primary and secondary carbon atoms). The formation of trimethylsilyl (TMS) ethers is not influenced by structural molecular differences when only hydroxy groups are involved in derivatization. Combined trimethylsilylation and acylation showed that compounds with a secondary carbon atom linked to the nitrogen atom form mainly N-TFA-O-TMS derivatives, with a small amount of N-TMS-O-TMS derivatives. Compounds with tert-butyl substituents at the amino group (bambuterol, salbutamol and terbutaline) formed O-TMS derivatives as the main products, although a limited amount of trifluoroacylation at the nitrogen atom also occurred. Cyclic methylboronates were formed with bambuterol, clenbuterol, formoterol, salbutamol and salmeterol. Owing to hydroxy substituents in unsuitable positions for ring formation, this procedure was not effective for fenoterol and terbutaline. Mass spectra of different derivatives and tentative fragmentation profiles are also shown. For screening purpose (e.g. sports drug testing), derivatization with MSTFA or BSTFA alone is recommended as a comprehensive derivatization technique for beta(2)-agonists owing to minimal by-product formation; formation of cyclic methylboronates can be useful for confirmation purposes. Detection limits were obtained for the TMS and cyclic methylboronate derivatives using the derivatizing reagents MSTFA and trimethylboroxine, respectively. For most of the compounds, lower detection limits were found for the TMS derivatives., (Copyright 2000 John Wiley & Sons, Ltd.)

A designed peptide, PGAa showed an excellent antifungal activity as well as an efficient bactericidal activity toward gram-positive, especially in the pathogenic yeast Candida albicans 28838. The solution structures of PGAa have been determined both in 40% TFE/water solution and DPC micelle by CD and NMR spectroscopy. Based on NOEs, vicinal coupling constants, backbone amide exchange rates, and chemical shift indices, PGAa formed a long amphipathic alpha-helical conformation in both TFE and DPC micelle environments, spanning the residues Ile(2)-Ala(19) in TFE and Lys(5)-Ala(19) in DPC micelle, respectively. Solution structures suggested that the hydrophobic residues would interact with the fatty acyl chains of the lipid bilayer, while the positively charged side-chains exposed to aqueous environments. Therefore, we conclude that the alpha-helical structure as well as the highly amphiphatic nature of PGAa peptide may play a critical role in its antimicrobial activity as well as selectivities in different species., (Copyright 2000 Academic Press.)

Trophozoites of the parasitic protozoa, Entamoeba histolytica, synthesize a cell surface lipoglycoconjugate, termed lipophosphoglycan, which is thought to be an important virulence factor and potential vaccine candidate against invasive amebiasis. Here, we show that the E. histolytica lipophosphoglycans are in fact glycosylphosphatidylinositol (GPI)-anchored proteophosphoglycans (PPGs). These PPGs contain a highly acidic polypeptide component which is rich in Asp, Glu and phosphoserine residues. This polypeptide component is extensively modified with linear glycan chains having the general structure, [Glcalpha1-6](n)Glcbeta1-6Gal (where n=2-23). These glycan chains can be released after mild-acid hydrolysis with trifluoroacetic or hydrofluoric acid and are probably attached to phosphoserine residues in the polypeptide backbone. The PPGs are further modified with a GPI anchor which differs from all other eukaryotic GPI anchors so far characterized in containing a glycan core with the structure, Gal(1)Man(2)GlcN-myo-inositol, and in being heterogeneously modified with chains of alpha-galactose. Trophozoites of the pathogenic HM-1:IMSS strain synthesize two distinct classes of PPG which have polydisperse molecular masses of 50-180 kDa (PPG-1) and 35-60 kDa (PPG-2) and are modified with glucan side-chains of different average lengths. In contrast, the non-pathogenic Rahman strain synthesizes one class of PPG which is only elaborated with short disaccharide side-chains (i.e. Glcbeta1-6Gal). However, the PPGs are abundant in all strains (8x10(7) copies per cell) and are likely to form a protective surface coat., (Copyright 2000 Academic Press.)

The kinetics of reaction between trifluoroacetic acid as an acid of medium strength and the carotenoids beta-carotene, zeaxanthin, canthaxanthin, and astaxanthin has been examined in detail including the effects of dioxygen, acid concentration, and carotenoid structure. Reaction between acid and carotenoid leads to species absorbing in the red and near-infrared (NIR) spectral regions, intermediates that subsequently disappear. ESR experiments clearly show that these species are not carotenoid radicals, although their NIR absorption is similar to the absorption of carotenoid radical cations. Under most reaction conditions, the disappearance of carotenoids follows pseudo-zero-order kinetics, whereas the reaction order is >1 with respect to acid, and the long-lived (hours) intermediates are suggested to be mono- (700 nm) and diprotonated carotenoid ( approximately 950 nm). Acid induces cis/trans-isomerization via the protonated intermediates, which also decay to nonradical species with shorter conjugated systems-most probably carotenoid esters. Slow protonization of the methine carbon is the primary step in the degradation, but dioxygen increases the rate as a result of formation of a charge-transfer complex with the carotenoids as indicated by a red-shift of the NIR absorption bands. Carotenoids with carbonyl groups (astaxanthin and canthaxanthin) have slower rates of degradation than beta-carotene and zeaxanthin, indicating preferential nondegradative protonation of the carbonyl groups.

Human amylin is the primary component of amyloid deposits found in the pancreatic beta-cells of patients with type 2 diabetes mellitus. Recently, two fragments of amylin have been identified in vivo. One fragment contains residues 17 to 37 of human amylin (AMYLIN17-37) and the other contains residues 24 to 37 (AMYLIN24-37). The secondary structure and amyloid forming ability of each peptide was determined at pH 5.5(+/-0.3) and pH 7.4(+/-0.3). Results at these two values of pH were very similar. Both peptides are predominantly unstructured in solution (CD) but adopt a significant amount of beta-sheet secondary structure upon aggregation (FTIR). Transmission electron microscopy (TEM) confirmed the presence of amyloid fibrils. AMYLIN24-37 was further dissected by studying peptides corresponding to residues 24 to 29 and 30 to 37. The AMYLIN30-37 peptide forms amyloid deposits. Samples of the 24 to 29 fragment which had TFA as the associated counterion formed ordered deposits but samples associated with HCl did not. Residues 20 to 29 are traditionally thought to be the amyloidogenic region of amylin, but this study demonstrates that peptides derived from other regions of amylin are capable of forming amyloid, and hence indicates that these regions of amylin can play a role in amyloid formation., (Copyright 1999 Academic Press.)

Skeletal muscle contraction and relaxation is efficiently modulated through the reaction of reactive oxygen-nitrogen species with sarcoplasmic reticulum protein thiols in vivo. However, the exact locations of functionally important modifications are at present unknown. Here, we determine by HPLC-MS that the modification of one (out of 24) Cys residue of the sarcoplasmic reticulum (SR) Ca-ATPase isoform SERCA1, Cys(349), by peroxynitrite is sufficient for the modulation of enzyme activity. Despite the size and nature of the SR Ca-ATPase, a 110 kDa membrane protein, identification and quantitation of Cys modification was achieved through labeling with 4-(dimethylamino)phenylazophenyl-4'-maleimide (DABMI) and/or N-(2-iodoethyl)trifluoroacetamide (IE-TFA) followed by an exhaustive tryptic digestion and on-line HPLC-UV-electrospray MS analysis. The reaction with IE-TFA generates aminoethylcysteine, a new trypsin cleavage site, which allows the production of specific peptide fragments that are diagnostic for IE-TFA labeling, conveniently identified by mass spectrometry. Exposure of the SR Ca-ATPase to low concentrations (0.1 mM) of peroxynitrite resulted in the fully reversible chemical modification of Cys at positions 344, 349, 471, 498, 525, and 614 (nitrosylation of Cys(344) and Cys(349) was seen), whereas higher concentrations of peroxynitrite (0.45 mM) additionally affected Cys residues at positions 636, 670, and 674. When the SR Ca-ATPase was exposed to 0.45 mM peroxynitrite in the presence of 5.0 mM glutathione (GSH), thiol modification became partially reversible and S-glutathiolation was detected for Cys residues at positions 344, 349, 364, 498, 525, and 614. The extent of enzyme inactivation (determined previously) quantitatively correlated with the loss of labeling efficiency (i) of a single Cys residue and (ii) of the tryptic fragment containing both Cys(344) and Cys(349). Earlier results had shown that the independent selective modification of Cys(344) is functionally insignificant [Kawakita, M., and Yamashita, T. (1987) J. Biochem. (Tokyo) 102, 103-109]. Thus, we conclude that modification of only Cys(349) is responsible for the modulation of the SR Ca-ATPase activity by peroxynitrite.

A new sensitive and convenient method for the structural analysis of oligosaccharides was developed, in which partial acid hydrolysis was combined with two-dimensional sugar mapping of pyridylamino (PA)-oligosaccharides. A PA-oligosaccharide was partially hydrolyzed, the acid hydrolysis conditions being optimized so as to obtain various PA-oligosaccharide fragments with high yields from different types of PA-oligosaccharides. The acid hydrolyzates were then fractionated every 1 glucose unit by size-fractionation HPLC, and each fraction was analyzed by reversed-phase HPLC. The structure of each PA-oligosaccharide fragment was identified on a two-dimensional sugar map prepared with standard PA-sugar chains, after which the original PA-oligosaccharide was reconstructed from the reducing end terminal based on the obtained structures of the PA-oligosaccharide fragments. The method was successfully applied to resolving the structure of an N-linked sugar chain.

E-9 cells, a HepG2 cell line that has the alcohol-inducible human cytochrome P-4502E1 (CYP2E1) cloned into its genome, was tested for its ability to produce trifluoroacetyl (TFA) adducts from the metabolism of halothane. The metabolism of halothane results in the production of TFA halide that can readily react with cellular proteins to form TFA adducts, which can be detected by antibodies directed against them. E-9 cells formed TFA adducts when incubated with halothane. The major consistent bands that were detected were of molecular weights of approximately 50, 78, and 100 kDa. The formation of these adducts was dependent on halothane concentration and time of incubation with halothane. MV-5 cells, the control cell line that has the viral vector without the P-450, did not produce any adducts. Inhibitors of CYP2E1 function, such as 4-methylpyrazole, inhibited adduct formation. Furthermore, phorbol esters, which have been shown to increase the CYP2E1 level in this cell line, increased TFA adduct formation. This HepG2 cell line may be of value in studying the metabolism and toxicity of halothane in a human cell culture model.

Background: Hydrochlorofluorocarbons (HCFCs) are used increasingly in industry as substitutes for ozone-depleting chlorofluorocarbons (CFCs). Limited studies in animals indicate potential hepatotoxicity of some of these compounds. We investigated an epidemic of liver disease in nine industrial workers who had had repeated accidental exposure to a mixture of 1,1-dichloro-2,2,2-trifluoroethane (HCFC 123) and 1-chloro-1,2,2,2-tetrafluoroethane (HCFC 124). All nine exposed workers were affected to various degrees. Both compounds are metabolised in the same way as 1-bromo-1-chloro-2,2,2-trifluoroethane (halothane) to form reactive trifluoroacetyl halide intermediates, which have been implicated in the hepatotoxicity of halothane. We aimed to test whether HCFCs 123 and 124 can result in serious liver disease., Methods: For one severely affected worker liver biopsy and immunohistochemical stainings for the presence of trifluoroacetyl protein adducts were done. The serum of six affected workers and five controls was tested for autoantibodies that react with human liver cytochrome-P450 2E1 (P450 2E1) and P58 protein disulphide isomerase isoform (P58)., Findings: The liver biopsy sample showed hepatocellular necrosis which was prominent in perivenular zone three and extended focally from portal tracts to portal tracts and centrilobular areas (bridging necrosis). Trifluoroacetyl-adducted proteins were detected in surviving hepatocytes. Autoantibodies against P450 2E1 or P58, previously associated with halothane hepatitis, were detected in the serum of five affected workers., Interpretation: Repeated exposure of human beings to HCFCs 123 and 124 can result in serious liver injury in a large proportion of the exposed population. Although the exact mechanism of hepatotoxicity of these agents is not known, the results suggest that trifluoroacetyl-altered liver proteins are involved. In view of the potentially widespread use of these compounds, there is an urgent need to develop safer alternatives.

The anesthetic, halothane, is bioactivated by the liver cytochrome P450 system to trifluoroacetyl-chloride, which can readily acylate liver protein. Covalent binding of the trifluoroacetyl moiety may result in hapten formation leading to the induction of an immune response and ultimately halothane hepatitis. In this study the presence of trifluoroacetylated-protein adducts in Kupffer cells was investigated to learn how the immune system might come in contact with the proteins. Guinea pigs were exposed to 1.0% halothane, 40% oxygen for 4 h. Kupffer cells were isolated on days 1 through 9 post-exposure, by liver perfusion and purification by elutriation. Using gel electrophoresis and Western blotting techniques, it has been demonstrated that Kupffer cells obtained from halothane-treated guinea pigs, do carry trifluoroacetyl-protein adducts as recognized by an anti-trifluoroacetyl-rabbit serum albumin antibody. Apparent molecular weights of polypeptides bound by trifluoroacetyl were of a wide range, 25-152 kDa. Bands were most prominent in the larger Kupffer cells with more appearing at lower molecular weights. Trifluoroacetyl-protein adducts were not detected in lung, spleen, lymph node or peripheral blood macrophages. This work suggests a role for Kupffer cells in the presentation of altered proteins in the liver to cells of the immune system.

Although trifluoroacetate (TFA), a breakdown product of chlorofluorocarbon replacements, is being dispersed widely within the biosphere, its ecological fate is largely unknown. TFA was added experimentally to an upland, northern hardwood forest and to a small forest wetland ecosystem within the Hubbard Brook Experimental Forest in New Hampshire. Inputs of TFA were not transported conservatively through these ecosystems; instead, significant amounts of TFA were retained within the vegetation and soil compartments. More TFA was retained by the wetland ecosystem than by the upland forest ecosystem. Using simulation modeling, TFA concentrations were predicted for soil and drainage water until the year 2040.

Nod factors are secreted lipo-oligosaccharides produced by symbiotic nitrogen-fixing Rhizobium bacteria that induce nodule formation on the roots of host leguminous plants. Two biologically active fractions (NodNGRA and NodNGRB) were isolated by reversed-phase HPLC from the culture supernatant of a Nod factor overproducing strain of Rhizobium sp. NGR234. NodNGRA and NodNGRB are heterogeneous mixtures of N-acylated 2-O-methylfucosylated chitomers, in which the fucosyl residue may be either 3-sulfated (NodNGRA), or 4-O-acetylated or nonsubstituted (NodNGRB). Structurally analogous series of compounds occur with either N-vaccenic (C18:1) or N-palmitic (C16:0) substituents. The presence of 6-O-carbamoyl groups on the GlcNMe-Acyl residue occurs on some molecules, while others are di-O-carbamoylated. Detailed structural analysis of seventeen Nod factors are reported here.

Arachidonic acid was oxidized by iron ascorbate. Samples were withdrawn in time intervals. The aldehydic oxidation products were trapped by preparation of pentafluorbenzyloximes. Their trimethylsilylated derivatives were subjected to analysis by GC/MS. The main aldehydic lipid peroxidation product was found to be the well-known 4-hydroxy-2-nonenal (HNE), but 2-hydroxy heptanal (HH) -- a previously unknown lipid peroxidation product of arachidonic acid -- was detected to be nearly equally abundant. Malondialdehyde (MDA), glyoxal and 2-hydroxy-4-decenal (HDE) were detected to be produced in up to 100 times lower amounts compared to HNE. The amounts of aldehydes increased steadily with time. In addition, n-l-hydroxy-n-oxo acids were detected. Similar aldehydes were obtained by iron ascorbate-induced oxidation of hydroxy acids derived by NaBH4-reduction of 13-hydroperoxy-9-cis-11-trans-octadecadienoic acid. Since this and analogous hydroxy acids (LOHs) are the main biological degradation products of hydroperoxides of unsaturated acids (LOOHs) their further peroxidation seems to be a main source of toxic aldehydes.

The effects of isoflurane, 1-chloro-2,2,2-trifluoroethyl difluoromethyl ether, on the oxidative metabolism of halothane to produce trifluoroacetic acid (TFA) and on the reductive metabolism of halothane to produce chlorodifluoroethylene (CDE) and chlorotrifluoroethane (CTE) in liver microsomes of guinea pig were examined. Isoflurane enhanced the production of CDE and CTE and inhibited the production of TFA. Isoflurane enhanced cytochrome P450 reduction and formation of an intermediate complex with cytochrome P450 without enhancement of NADPH-cytochrome P450 reductase (EC 1.6.2.4) activity. We conclude that isoflurane interacts with cytochrome P450 to prevent the formation of the halothane-cytochrome P450 complex, causing inhibition of the oxidative dehalogenation. This interaction of isoflurane enhances the reduction of cytochrome P450 and the formation of a reductive intermediate-cytochrome P450 complex under anaerobic conditions causing reductive dehalogenation of halothane.

In this study we have investigated the mechanism of the processing of trifluoroacetylated liver microsomal protein antigens associated with halothane hepatitis to learn how the immune system might come in contact with these proteins to form antibodies directed against them. Rats were treated with halothane and parenchymal (PC) and non-parenchymal cells (NPC) were isolated 16 hours later. Immunoblotting of the cell lysates with antisera directed against the trifluoroacetyl hapten showed the presence of high levels of trifluoroacetylated proteins in parenchymal cells, whereas none of these proteins were detected in endothelial or Kupffer cells that were isolated by centrifugal elutriation. The half-lives of 100-, 82-, 80-, 63-, 59-, 58-, and 57-kd trifluoroacetylated and native carrier proteins of the trifluoroacetyl hapten in cultures of rat primary parenchymal cells were approximately 1 day. The turnovers of all of these trifluoroacetylated proteins, except for that of the trifluoroacetylated 100-kd protein, were inhibited by treatment of the cells with ammonium chloride, leupeptin, 4-(2-aminoethyl)-benzenesulfonyl fluoride, or 3-methyl-adenine (3-MA). These results indicate that, in liver, the major source of the formation of trifluoroacetylated antigens associated with halothane hepatitis is the parenchymal cells, It appears that most of the trifluoroacetylated antigens and possibly the native carrier protein of the trifluoroacetyl haptens are transferred from the endoplasmic reticulum (ER) to an acidic compartment of PCs, where they are enzymatically degraded. The processing of the trifluoroacetylated proteins by this pathway may be a protective mechanism that prevents these covalently altered proteins from inducing an antibody response in most patients who are administered halothane.

Halothane hepatitis appears to result from an inappropriate immune response to the products of halothane metabolism. Attempts to produce an animal model for halothane hepatitis have been largely unsuccessful. Although guinea pigs produce neoantigens following treatment with halothane, the subsequent antibody response is weak, possibly accounting for the failure to produce halothane hepatitis in these animals. In order to increase the antibody response to halothane neoantigens, three methods for trifluoroacetylating proteins were used. Guinea pigs were either treated with S-ethylthiotrifluoroacetate, autologous lymphocytes trifluoroacetylated ex vivo, or immunized with trifluoroacetylated mycobacterial protein, followed by exposure to halothane, and examined for anti-halothane metabolite antibodies (anti-TFA antibodies). Animals treated with S-ethylthiotrifluoroacetate developed anti-TFA antibodies, and following exposure to halothane exhibited an enhanced antibody response. Treatment with trifluoroacetylated lymphocytes also resulted in an enhanced anti-TFA antibody response following halothane exposure. Immunization with trifluoroacetylated mycobacterial proteins resulted in very high anti-TFA antibody titers. However, subsequent exposure to halothane had no observable effect on specific antibody titers. Exposure to halothane, regardless of treatment, resulted in the production of anti-microsomal protein antibodies. Signs of halothane hepatitis were not observed, indicating that enhancement of the humoral immune response does not appear to be sufficient for production of halothane hepatitis.

Previous studies have shown that exposure of phenobarbital-pretreated rats to halothane in 10% O2 causes centrilobular necrosis, induces expression of the 72-kDa heat shock protein (HSP72), and produces several trifluoroacetylated adducts. In the present study the time course of development of the centrilobular lesion, as measured by histochemistry, was compared with the time course of appearance of both trifluoroacetylated adducts and HSP72, as measured by Western blotting. One group of 20 rats was pretreated with phenobarbital for 5 days, whereas a second group of two rats was left as untreated controls. Ten phenobarbital-pretreated rats were exposed for 2 hr to 1% halothane in 10% O2 and 10 were exposed to 1% halothane in 20% O2. At either 2, 4, 6, or 24 hr after exposure, livers were excised and frozen without fixation. Thin sections stained with hematoxylin and eosin demonstrated that centrilobular lesions occurred at 6 hr and became extensive at 24 hr in rats pretreated with phenobarbital and exposed to 1% halothane in 10% O2. The time course of appearance of both trifluoroacetylated adducts and HSP72 was determined by Western blotting. Trifluoroacetylated adducts appeared in all rats exposed to halothane by 2 hr, lasted until 6 hr, and then diminished by 24 hr. In contrast, HSP72 was induced only in the rats pretreated with phenobarbital and exposed to 1% halothane in 10% O2. HSP72 appeared in both the nuclear and supernatant fractions at 6 hr after exposure and was intense 24 hr after exposure.

1. Chlorofluorohydrocarbons are presently being developed as alternatives for ozone-depleting chlorofluorocarbons. 1,1-Dichloro-2,2,2-trifluoro-[2-14C]-ethane (HCFC-123) is a chlorofluorohydrocarbon with potential widespread use and associated human exposure. As a part of the toxicological evaluation of HCFC-123, its metabolism was studied in rodents in a closed recirculating exposure system. 2. Two male rats were individually exposed for 6 h. Excretion of radioactivity was monitored for 48 h after the start of the exposure. Of the radioactivity introduced into the chamber, 14% was recovered in urine within the period of observation. Excretion of metabolites in the urine was very slow. 3. Trifluoroacetic acid was the major metabolite of HCFC-123 and N-trifluoroacetyl-2-aminoethanol and N-acetyl-S-(2,2-dichloro-1,1-difluoroethyl)-L-cysteine were identified as minor urinary metabolites of HCFC-123. 4. Forty-eight hours after the start of the exposure, covalent binding of radioactive metabolites to protein was highest in liver followed by kidney and lung. Covalent binding above background levels was not observed in pancreas and testis, the target organs of HCFC-123 tumourigenicity. 5. These results suggest that the biotransformation of HCFC-123 in rodents follows a pathway identical to those of the extensively studied structural analogue halothane.

The transport of trifluoroacetate (TFA) and difluorophosphate (DFP) into and out of human and sheep erythrocytes was measured using 19F-NMR. The pathways for the transport in human erythrocytes were characterised by differentiating between the transport inhibition caused by different reagents. (1) Pre-treatment of human erythrocytes with N-ethylmaleimide (10 mM) caused a decrease of the membrane-permeability coefficients for TFA influx and efflux to 0.74 +/- 0.05 and 0.83 +/- 0.09-times, respectively, of those determined in the absence of inhibition. Concomitantly there was no apparent effect on the band-3-mediated transport of DFP. Thus, the decrease of the permeability of TFA is consistent with the inhibition being that of the monocarboxylate transporter. (2) Inhibition of TFA and DFP exchange was also seen in human erythrocytes treated with p-chloromercuriphenylsulfonate (pCMBS). The extent of inhibition reached a maximum value for the pCMBS concentrations beyond which further inhibition was not achieved and there was substantial residual exchange of the two solutes. (3) Residual flux of TFA was found in the presence of high concentrations of the inhibitors, alpha-cyano-4-hydroxycinnamate (> or = 4 mM) or 4,4'-dinitrostilbene-2,2'-disulfonate (> or = 1 mM) when each compound was used alone. (4) Complete inhibition of TFA uptake was obtained when human erythrocytes were treated with both alpha-cyano-4-hydroxycinnamate (4 mM) and a stilbene disulfonate. It was, therefore, concluded that simple diffusion of TFA via the lipid bilayer was negligible in human erythrocytes and that incomplete inhibition of the monocarboxylate transporter occurred when the compounds were used alone.

A monospecific antibody (anti-CF3CO antibody) was obtained by affinity chromatography on a N epsilon-trifluoroacetyl-L-lysine (CF3CO-Lys) matrix of a rabbit polyclonal antiserum, directed against trifluoroacetylated protein adducts (CF3CO-proteins). The anti-CF3CO antibody recognized distinct CF3CO-proteins on immunoblots of a liver biopsy obtained from a human individual 10 h after halothane anaesthesia. Cross-reactive proteins of 52 kDa and 64 kDa were recognized on immunoblots of livers obtained from human individuals not exposed to halothane. Recognition of both CF3CO-proteins and the 52-kDa and 64-kDa cross-reactive proteins was abolished in the presence of 1 mM CF3CO-Lys. Anti-CF3CO antibody, affinity-adsorbed to the 52-kDa or the 64-kDa cross-reactive proteins of human liver, recognized the majority of target CF3CO-proteins on immunoblots of the human liver biopsy of an individual exposed to halothane. Liver biopsies of 5 out of 7 (71%) patients with halothane hepatitis exhibited an absence or low amounts of immunorecognizable 52-kDa and/or 64-kDa cross-reactive proteins. In contrast, of 22 control human individuals tested, all liver tissue samples were positive for the 52-kDa and/or the 64-kDa cross-reactive proteins. These data indicate that epitopes on the cross-reactive proteins of 52 kDa and 64 kDa of human liver bear strong immunochemical resemblance to epitopes on human liver CF3CO-proteins. Low-level expression of the cross-reactive proteins of 52 kDa and 64 kDa is discussed as one possible factor in human susceptibility to halothane hepatitis.

A general procedure is presented for the isolation of several liver microsomal target proteins of the reactive trifluoroacetyl halide metabolite of halothane. It was found that most of these proteins could be selectively extracted from microsomes with 0.1% sodium deoxycholate and separated into partially purified fractions by DEAE-Sepharose anion-exchange chromatography. Using this method, we describe the isolation and identification of a 63-kDa target protein of halothane in rat liver. Amino acid sequences of the N-terminal and of several internal peptides of the protein, as well as the deduced amino acid sequence of a nearly full-length rat liver cDNA clone of the protein, showed 98% identity with a reported murine cDNA that encodes for calreticulin, a major calcium-binding protein of the lumen of endoplasmic reticulum. Although it remains to be determined what role calreticulin has in the development of halothane hepatitis, this study has shown that calreticulin can be a target of reactive metabolites of xenobiotics.

As part of a program directed towards the chemical syntheses of potential metabolites and analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism, several routes have been explored for the preparation of 3 beta-hydroxy-15-keto-5 alpha-chol-8(14)-en-24-oic acid (IV). These investigations led to a remarkably specific and efficient side-chain oxidation of I. For example, treatment of the acetate of I with a mixture of trifluoroacetic anhydride, hydrogen peroxide, and sulfuric acid for 3.5 h at -2 degrees C gave a crude product consisting of 3 beta-acetoxy-24-trifluoroacetoxy-5 alpha-chol-8(14)-en-15-one (XI), 3 beta-acetoxy-24-hydroxy-5 alpha-chol-8(14)-en-15-one (XII), and 3 beta, 24-diacetoxy-5 alpha-chol-8(14)-en-15-one (XIII) in yields of 58%, 8%, and 3%, respectively, by HPLC analysis. XI was readily hydrolyzed to XII upon treatment with triethylamine in methanol at room temperature. Oxidation of XII with Jones reagent gave 3 beta-acetoxy-15-keto-5 alpha-chol-8(14)-en-24-oic acid (XVIII) from which its methyl ester (IX) was prepared by treatment with diazomethane. Mild alkaline hydrolysis of XVIII gave the 3 beta-hydroxy-delta 8(14)-15-keto C24 acid (IV). Hydrolysis of the crude product of the side-chain oxidation with K2CO3 in methanol gave 3 beta,24-dihydroxy-5 alpha-chol-8(14)-en-15-one (XIV) which was oxidized with Jones reagent to yield 3,15-diketo-5 alpha-chol-8(14)-en-24-oic acid (XV). Treatment of XV with diazomethane gave its methyl ester (XVI) which, upon controlled reduction with NaBH4, yielded methyl 3 beta-hydroxy-15-keto-5 alpha-chol-8(14)-en-24-oate (XVII). Compound IX was also prepared by an independent route. Full 1H and 13C NMR assignments are presented for 12 new compounds. IV caused a approximately 56% reduction of the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells at a concentration of 2.5 microM. In contrast, the corresponding 3,15-diketo acid XV had no detectable effect on reductase activity under the same conditions.

A monoform antibody [anti-TFA antibody] against TFA-protein adducts (TFA-adducts) was obtained by affinity purification of a polyclonal antiserum, raised in rabbits against TFA-rabbit serum albumin, on a N-epsilon-TFA-L-lysine matrix coupled to Affi-Gel 102. The anti-TFA antibody did recognize TFA-adducts of distinct molecular mass on Western blots of hepatocyte homogenates or microsomal membranes obtained from rats pretreated with halothane. The anti-TFA antibody also recognized cross-reactive polypeptides with apparent molecular masses of 52 kDa and 64 kDa on Western blots of hepatocyte homogenates obtained from rats not treated with halothane or metabolites thereof. The 52-kDa and 64-kDa cross-reactive polypeptides were localized in the 3,000 x g particulate fraction of liver homogenates. Recognition, on Western blots, of TFA-adducts and both the 52-kDa and 64-kDa cross-reactive polypeptides by anti-TFA antibody was sensitive to competition by N-epsilon-TFA-L-lysine (IC50 less than 100 microM) and N-epsilon-acetyl-L-lysine (IC50 approximately 10 mM). Treatment with piperidine (1 M) did abolish the recognition of TFA-adducts but not that of the 52-kDa and the 64-kDa cross-reactive polypeptides by anti-TFA antibody on Western blots. In antibody-exchange experiments, anti-TFA antibody was affinity-adsorbed on Western blots to the 52-kDa or the 64-kDa cross-reactive polypeptides of the rat heart, followed by spontaneous transfer to target TFA-adducts present on Western blots of rat liver microsomal membranes. The majority of these target TFA-adducts were recognized by anti-TFA antibody transferring from the source 52-kDa or 64-kDa cross-reactive polypeptides. When examined up to 10 days after exposure of rats to a single dose of halothane, no influence on the constitutive level of expression, in the liver, of either cross-reactive polypeptide was observed. In contrast, TFA-adducts were persistent for greater than 90 hr but less than 10 days. In addition to the liver, the 52-kDa and the 64-kDa cross-reactive polypeptides were prominently expressed in the heart and the kidney and, to a much lesser degree, in the spleen, the thymus, the lung, and skeletal muscle of the rat. Considerable variation in the level of expression of the 52-kDa and the 64-kDa cross-reactive polypeptides was recognized in livers of the six human individuals tested so far.(ABSTRACT TRUNCATED AT 250 WORDS)

The delayed fulminant form of halothane hepatotoxicity is thought to be triggered by an immune response to haptenic adducts formed by a metabolite, trifluoroacetyl chloride. In this study we demonstrate that antibodies purified from the sera of rabbits sensitized to a trifluoroacetyl-protein adduct will cross-react with a trifluoroacetyl-phosphatidylethanolamine adduct. Trifluoroacetyl adducts of both rabbit serum albumin (TFA-RSA) and dioleoylphosphatidylethanolamine (TFA-DOPE) were prepared. The TFA-RSA was coupled to an Affigel-10 affinity column to purify hapten-selective immunoglobulin (Ig) G antibodies (anti-TFA-RSA IgG) from the sera of rabbits given i.m. injections of TFA-RSA. The TFA-DOPE was purified by high-performance liquid chromatography and the structure confirmed with direct chemical ionization mass spectrometry. Lamellar liposomes containing a mixture of 5% TFA-DOPE, 71% DOPE and 24% dioleoyl-phosphatidylcholine, as well as hexagonal phase micelles containing 5% TFA-DOPE and 95% DOPE, were prepared by sonication. Anti-TFA-RSA IgG antibodies were added to each of these lipid mixtures for 30 min, fluorescein-conjugated goat-antirabbit IgG antibodies were added next for an additional 30 min and then binding of anti-TFA-RSA IgG antibodies to TFA-DOPE was quantified by flow cytometry. Anti-TFA-RSA IgG antibodies bound to TFA-DOPE only when it was incorporated into hexagonal phase micelles. These findings suggest that TFA-phosphatidylethanolamine adducts that reside in nonlamellar domains on the hepatocyte surface could be recognition sites for anti-TFA-adduct antibodies and potentially participate in immune-mediated hepatotoxicity.

The formation of trifluoroacetic acid (TFAA) from halothane under aerobic conditions and that of chlorotrifluoroethane (CTE) and chlorodifluoroethylene (CDE) from halothane under anaerobic conditions were studied using guinea pig liver microsomes. The formation of TFAA was inhibited by specific inhibitors of cytochrome P450 (P450), such as carbon monoxide and metyrapone and was dependent upon P450 contents. The maximum activity of the TFAA formation was obtained at pH 6.0. On the other hand, the maximum activity to form CTE and CDE was obtained at pH 7.4. The formation of TFAA reached a plateau at a halothane concentration above 0.17 mM, but the rate of formation of CDE and CTE was dependent upon a halothane concentration up to 1.5 mM. The values of apparent Michaelis-Menten constant (Km) and maximum velocity (Vmax) for TFAA formation were 0.067 mM and 0.349 nmol/nmol P450/min respectively, those for CDE formation were 0.983 mM and 0.326 nmol/nmol P450/min respectively, and those for CTE formation were 1.71 mM and 0.752 nmol/nmol P450/min respectively. These results showed clearly that the formation of TFAA, CDE and CTE was catalyzed by the P450 system in guinea pig liver microsomes. Under optimal conditions, saturation was observed in the formation of TFAA from halothane at a halothane concentration above 0.17 mM but the formation of CDE and CTE was not saturated at this concentration, and the value of apparent Km for TFAA formation was lower than those for CDE and CTE formation.

Previous studies have shown that antibodies in the sera of halothane hepatitis patients recognize trifluoroacetylated liver microsomal proteins (neoantigens) of 100 kDa, 76 kDa, 59 kDa, 57 kDa, and 54 kDa. In the present investigation, factors that might affect the level of expression of the neoantigens were investigated. A study of the time course of neoantigen expression in halothane-treated rats revealed that the 100 kDa, 76 kDa, 59 kDa, and 57 kDa neoantigens were longer-lived than the 54 kDa neoantigen and could be detected in the liver up to a week after the administration of halothane. Pretreatment of rats with isoniazid, which is known to induce cytochrome P-450 IIE1, appeared to increase the expression of each of the neoantigens, whereas inducers of several other forms of cytochrome P-450 had either very little effect or decreased the expression of several of the neoantigens. Female rats appeared to express some of the neoantigens at a higher level than that found in males. Examination of the organ distribution of the trifluoroacetylated neoantigens showed that, of the tissues examined, only the liver contained appreciable levels of the neoantigens. These results indicate that the level of expression and possibly the immunogenicity of the trifluoroacetylated liver neoantigens may be influenced by their half-lives and the repertoire of cytochrome P-450 present in the liver.

The biotransformation of halothane was studied using liver slices. Precision-cut Hartley male guinea pig liver slices (1 cm diameter; 250-300 microns thick) were incubated in sealed roller vials containing supplemented Krebs-Henseleit buffer at 37 degrees C under different O2 tensions (2.5, 21, and 95%). After a 1-hr preincubation, halothane was vaporized in the vial producing a 1.9 mM medium concentration. Halothane metabolites (Br-, trifluoroacetic acid, F-) were measured at 2, 4, and 6 hr. Viability of the incubated slices was verified by determining intracellular K+ content and levels of cytochrome P-450, which were maintained under 95% O2 atmosphere but decreased with lower O2 tensions (2.5%). The highest fluoride production was 300 +/- 22 pmol/mg slice weight/6 hr at low O2 tension (2.5%). Defluorination decreased with increasing O2 tension to undetectable levels under 95% O2. Production of the oxidative metabolite, trifluoroacetic acid, was highest at 95% O2 (2.35 +/- 0.17 nmol/mg slice weight/6 hr). Trifluoroacetic acid production decreased with decreasing O2 tension. Br- production was the highest at 21% O2 (1.8 +/- 0.13 nmol/mg slice weight/6 hr). Production of Br- was not dependent on the O2 tension. The guinea pig slices are capable of biotransforming halothane (oxidative/reductive); therefore, this in vitro system appears suitable for studying the biotransformation of halothane.

The structure of a hepatotoxic peptide from the cyanobacterium Nodularia spumigena was determined using 1D and 2D proton nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. The toxin was a cyclic pentapeptide (mol. wt 824.5) with the structure cyclo-(beta-methylisoAsp-Arg-Adda-isoGlu-N-methylde hydrobutyric acid) (Adda: 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid).

Previous studies have demonstrated that patients with halothane-induced hepatitis have serum antibodies that are directed against novel liver microsomal neoantigens and have suggested that these neoantigens may play an immunopathological role in development of the patients' liver damage. These investigations have further revealed that the antibodies are directed against distinct polypeptide fractions (100 kDa, 76 kDa, 59 kDa, 57 kDa, 54 kDa) that have been covalently modified by the reactive trifluoroacetyl halide metabolite of halothane. In this paper, the trifluoroacetylated (TFA) 59-kDa neoantigen (59-kDa-TFA) recognized by the patients' antibodies was isolated from liver microsomes of halothane-treated rats by chromatography on an immunoaffinity column of anti-TFA IgG. Antibodies were raised against the 59-kDa-TFA protein and were used to purify the native protein from liver microsomes of untreated rats. Based upon its apparent monomeric molecular mass, NH2-terminal amino acid sequence, catalytic activity, and other physical properties, the protein has been identified as a previously characterized microsomal carboxylesterase (EC 3.1.1.1). A similar strategy may be used to purify and characterized neoantigens associated with other drug toxicities that are believed to have an immunopathological basis.