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.

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.

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.

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.

Hepatobiliary metabolism and excretion of adriamycin and N-trifluoroacetyladriamycin-14-valerate in the rat.

In connection with mechanism of action studies with N-trifluoroacetlyadriamycin-14-valerate (AD 32), a superior Adriamycin (ADR) analog under development in these laboratories, serial bile samples were collected from male Sprague-Dawley rats given a single i.v. dose of either ADR (4 mg/kg) or AD 32 (20 mg/kg) and were analyzed for anthracyclines by thin-layer chromatography-fluorometry and high-performance liquid chromatography. For ADR, 20% of the administered dose was accounted for at 24 hr, whereas 80% of the AD 32 dose were excreted into the bile by this time. ADR underwent little biotransformation; 80% of the 48-hr cumulative fluorescence excretion was attributable to unchanged drug, one-half the remainder was adriamycinol, and the balance was polar conjugates. In contrast, AD 32 underwent extensive metabolism to N-trifluoracetyladriamycin, N-trifluoroacetyladriamycinol, and polar conjugates, mostly glucuronides of N-trifluoroacetyladriamycin and N-trifluoroacetyladriamycinol. Based on direct and indirect evidence, ADR was not a metabolite of AD 32.

The binding of N-trifluoroacetyl chito-oligosaccharides to wheat-germ agglutinin: a fluorescence investigation.

We describe the synthesis of N-trifluoroacetyl chito-oligosaccharides and their use as ligands to probe the binding sites of wheat-germ agglutinin, a lectin specific for N-acetylglucosamine. The binding is monitored using intrinsic protein fluorescence, which is due to tryptophan side-chains. We present arguments purporting to show the presence of a fluorophore close to each of the four sites. The binding of chito-oligosaccharides to wheat-germ agglutinin is complex and can only be approximately described by an independent and equivalent sites model. This model applies when the ligand concentration range is restricted to higher values. The possible role of ligand-mediated protein aggregation and of site inequivalence is discussed. We find that the affinity of trifluoroacetylated chito-oligosaccharides for wheat-germ agglutinin is higher than that of the N-acetylated parent compounds, the difference increasing with chain length. Our results are in agreement with a model of the binding site previously proposed by Clegg et al. (Biochemistry 22 (1983) 4797-4804).

Characterisation of erythrocyte transmembrane exchange of trifluoroacetate using 19F-NMR: evidence for transport via the monocarboxylate transporter.

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.

Intrinsic catalytic activity of procarboxypeptidase A. A kinetic study using fluorine analogues.

Bovine procarboxypeptidase A displays substantial catalytic activity toward halogenated acyl-amino acids, the most active of which is trifluoroacetyl-L-phenylalanine (TFAc-L-Phe). Though this activity is not as great as for the native enzyme, it is quite substantial and far beyond the range of adventitious activation. Both DL-benzylcuccinate and beta-phenylpropionate inhibit zymogen hydrolysis of TFAc-L-Phe, the former with a K1 of 4.1 micrometer and the latter, 900 micrometer (a value much higher than the corresponding enzyme). Apo procarboxypeptidase A will also hydrolyze TFAc-L-Phe, presumably the polarization of the carbonyl carbon being accomplished by the fluorine atoms in the absence of a specific metal ion. That this is not entirely the metal ion function is indicated by the fact that rate enhancements follow the order manganese procarboxypeptidase A approximately zinc procarboxypeptidase greater than apo-procarboxypeptidase. The results indicate considerable similarities for the zymogen-enzyme pair in terms of catalytic groups, pH dependence, specificity and the nature of their transition state binding sites. Some changes in the substrate or inhibitor binding sites are noted.

Analysis of amylin cleavage products provides new insights into the amyloidogenic region of human amylin.

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.

The major surface antigens of Entamoeba histolytica trophozoites are GPI-anchored proteophosphoglycans.

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.

Derivatization procedures for the detection of beta(2)-agonists by gas chromatographic/mass spectrometric analysis.

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.

Structure of a hepatotoxic pentapeptide from the cyanobacterium Nodularia spumigena.

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).

Isoflurane acts as an inhibitor of oxidative dehalogenation while acting as an accelerator of reductive dehalogenation of halothane in guinea pig liver microsomes.

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.

Effects of piperonyl butoxide on halothane hepatotoxicity and metabolism in the hyperthyroid rat.

A series of experiments were conducted to examine the potential role of phase I metabolism in halothane-induced liver injury in the hyperthyroid rat. The metabolism of halothane was determined in both hyperthyroid (triiodothyronine, 3 mg/kg per day, for 6 days) and euthyroid rats and in animals pre-treated with the cytochrome P-450 inhibitor piperonyl butoxide (75-100 mg/kg, i.p.). It was found that the hyperthyroid state, which is associated with a substantial increase in sensitivity to the hepatotoxic effects of halothane, decreases both oxidative and reductive routes of halothane metabolism in the rat. The production of trifluoroacetic acid (TFA), an oxidative metabolite, as well as that of chlorodifluoroethylene (CDF) and chlorotrifluoroethane (CTF), 2 reductive metabolites, was significantly reduced in hyperthyroid animals. Consistent with these findings serum and urinary bromide levels resulting from the formation of TFA, CDF or CTF were significantly reduced. The only route of halothane metabolism significantly increased by the hyperthyroid condition was the defluorination of halothane. Piperonyl butoxide administration did not render euthyroid animals sensitive to the halothane-induced hepatotoxicity and had no effect on the defluorination of halothane in euthyroid animals. However, piperonyl butoxide markedly increased the hepatotoxicity of halothane in hyperthyroid rats and, except for a modest increase in debromination reactions, decreased all measured indices of halothane metabolism including the defluorination of halothane. Thus, none of the observed changes in halothane metabolism produced by triiodothyronine or piperonyl butoxide treatment could be consistently correlated to the increases in hepatotoxicity linked to these 2 treatments. Based on these studies we suggest that the halothane hepatotoxicity induced in the hyperthyroid rat results from effects produced by either the parent compound or an as yet unidentified metabolite. In addition, these studies further demonstrate that considerable mechanistic differences exist for halothane-induced hepatotoxicity when comparing euthyroid and hyperthyroid animal models.

Kupffer cells from halothane-exposed guinea pigs carry trifluoroacetylated protein adducts.

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.

Previously unknown aldehydic lipid peroxidation compounds of arachidonic acid.

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.

Structural determination of symbiotic nodulation factors from the broad host-range Rhizobium species NGR234.

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.

Kinetics and mechanism of the primary steps of degradation of carotenoids by acid in homogeneous solution.

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.