Involvement of nitrergic neurons in colonic motility in a rat model of ulcerative colitis.

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.

Characterization of two carbonyl reductases from Ogataea polymorpha NBRC 0799.

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.

Raspberry Ketone Trifluoroacetate, a New Attractant for the Queensland Fruit Fly, Bactrocera Tryoni (Froggatt).

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.

Overexpression of a BAHD acyltransferase, OsAt10, alters rice cell wall hydroxycinnamic acid content and saccharification.

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.

Microbial degradation of pharmaceuticals followed by a simple HPLC-DAD method.

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.

Comparing inactivation protocols of Yersinia organisms for identification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

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.

Improved determination of D-glucosamine hydrochloride in health foods by HPLC using 7-fluoro-4-nitrobenzo-2-oxa-l,3-diazole as a derivative.

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.

From trifluoroacetate complex precursors to monodisperse rare-earth fluoride and oxyfluoride nanocrystals with diverse shapes through controlled fluorination in solution phase.

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.

Role of neutrophils in a mouse model of halothane-induced liver injury.

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.

Analysis of eluted peptides from type 1 diabetes-susceptible HLA class II molecules identified novel islet protein, heparin/heparan sulfate-interacting protein.

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.

Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates.

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.

Modulation of the antioxidant activity of HO* scavengers by albumin binding: a 19F-NMR study.

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.

Concordance between trifluoroacetic acid and hepatic protein trifluoroacetylation after disulfiram inhibition of halothane metabolism in rats.

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.

Effects of HCFC-123 exposure to maternal and infant rhesus monkeys on hepatic biochemistry, lactational parameters and postnatal growth.

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.

19F NMR of trifluoroacetyl-labeled cysteine mutants of myoglobin: structural probes of nitric oxide bound to the H93G cavity mutant.

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.

The fate and persistence of trifluoroacetic and chloroacetic acids in pond waters.

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.

Removal of N-terminal blocking groups from proteins.

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.

Modification of cysteine.

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.

In vivo 19F NMR chemical-shift imaging of Ancistrocladus species.

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.