Novel chemistry of abdominal defensive glands of nymphalid butterfly Agraulis vanillae.

Abdominal defensive glands of both sexes of the Gulf fritillary butterfly, Agraulis vanillae (Linnaeus) (Nymphalidae:Heliconiinae) emit a pronounced odor when disturbed. We have identified 6-methyl-5-hepten-2-one; oleic, palmitic, and stearic esters of the corresponding alcohol 6-methyl-5-hepten-2-ol; hexadecyl acetate; 1,16-hexadecanediol diacetate; and 1,15-hexade-canediol diacetate in the glandular exudate. Since we have determined that free-flying birds or birds in a butterfly conservatory discriminate against A. vanillae as prey, we suggest that the constituents in the glands may play a defensive role against potential avian predators.

Immunochemical evidence against the involvement of cysteine conjugate beta-lyase in compound A nephrotoxicity in rats.

BACKGROUND: Compound A, a degradation product of sevoflurane, causes renal corticomedullary necrosis in rats. Although the toxicity of this compound was originally hypothesized to result from the biotransformation of its cysteine conjugates into toxic thionoacyl halide metabolites by renal cysteine conjugate beta-lyase, recent evidence suggests that alternative mechanisms may be responsible for compound A nephrotoxicity. The aim of this study was to evaluate these issues by determining whether mercapturates and glutathione conjugates of compound A could produce renal corticomedullary necrosis in rats, similar to compound A, and whether renal covalent adducts of the thionacyl halide metabolite of compound A could be detected immunochemically. METHODS: Male Wistar rats were administered, intraperitoneally, N-acetylcysteine conjugates (mercapturates) of compound A (90 or 180 micromol/kg) or glutathione conjugates of compound A (180 micromol/kg) with or without intraperitoneal pretreatments with aminooxyacetic acid (500 micromol/kg) or acivicin (250 micromol/kg). Rats were killed after 24 h, and kidney tissues were analyzed for toxicity by histologic examination or for protein adducts by immunoblotting or immunohistochemical analysis, using antisera raised against the covalently bound thionoacyl halide metabolite of compound A. RESULTS: Mercapturates and glutathione conjugates of compound A both produced renal corticomedullary necrosis similar to that caused by compound A. Aminooxyacetic acid, an inhibitor of renal cysteine conjugate beta-lyase, did not inhibit the toxicity of the mercapturates, whereas acivicin, an inhibitor of gamma-glutamyltranspeptidase, potentiated the toxicity of both classes of conjugates. No immunochemical evidence for renal protein adducts of the thionacyl halide metabolite was found in rats 24 h after the administration of the mercapturates of compound A or in the kidneys of rats, obtained from a previous study, 5 and 24 h after the administration of compound A. CONCLUSION: The results of this study are consistent with the idea that a mechanism other than the renal cysteine conjugate beta-lyase pathway of metabolic activation is responsible for the nephrotoxicity of compound A and its glutathione and mercapturate conjugates in male Wistar rats.

The reaction of 4-hydroxy-2-nonenal with N alpha-acetyl-L-histidine.

The reaction of 4-hydroxy-2-nonenal and N alpha-acetyl-L-histidine was studied in pH 7.1 phosphate buffer. The main product isolated was assigned a cyclic hemiacetal structure formed by the addition of one of the imidazole nitrogen atoms to the alpha,beta-unsaturated aldehyde system of 4-hydroxy-2-nonenal. This structural assignment was based on the analyses of the NMR and mass spectral data of two derivatives obtained from the cyclic hemiacetal. The establishment of this cyclic hemiacetal structure supports the proposal made by Uchida and Stadtman6 that 4-hydroxy-2-nonenal modified histidyl residues in insulin by a Michael reaction.

Synthesis and properties of N-bromoacetyl-L-thyroxine.

A one-step bromoacetylation of L-thyroxine (T4) produces N-bromoacetyl-L-thyroxine (BrAcT4) in good yield. The reaction product is best purified by high-speed countercurrent chromatography. While HPLC is satisfactory only for purification of microgram and submicrogram quantities, amounts ranging from about 1 ng to 1 g of BrAcT4 can be processed by high-speed countercurrent chromatography (HSCCC), a method which we have previously used for the purification of N-bromoacetyl-3,3',5-triiodo-L-thyronine (BrAcT3). Operating conditions for the one-step synthesis of BrAcT4 and BrAcT3 differ due to differences in solubility and reactivity of the two hormones. BrAcT4 purified by HSCCC and shown to be pure by analytical HPLC has been characterized by alpha max and epsilon max in the near and far uv in several solvents, mass spectrum, 1H NMR spectrum, TLC in three solvent systems, retention time in reverse-phase HPLC (C18) in relation to the retention times of two internal standards, 3,3',5-triiodo-L-thyronine and T4, and melting point. Corresponding data for BrAcT3, not previously reported, have also been determined. The described procedure can provide not only substantial amounts of highly purified BrAcT4 for competition studies, but also 125I-labeled BrAcT4 of high specific activity for affinity labeling. Since solutions of BrAcT4 and of BrAcT3 undergo partial decomposition on evaporation to dryness, suitable procedures for the preparation of these hormones in solid form and for storage in solutions have been devised.

Synthesis and characterization of N-bromoacetyl-3,3',5-triiodo-L-thyronine.

N-Bromoacetyl-3,3',5-triiodo-L-thyronine and carrier-free [3'-125I]-N-bromoacetyl-3,3',5-triiodo-L-thyronine, to be used for affinity labeling of thyroid hormone receptors, were synthesized using a one-step procedure: a solution of the thyroid hormone 3,3',5-triiodo-L-thyronine and bromoacetyl bromide in ethyl acetate was refluxed for an optimal period of time which depends on the amount of hormone processed. The bromoacetylated hormone thus obtained was then fractionated by high-speed counter-current chromatography which yielded N-bromoacetyl-3,3',5-triiodo-L-thyronine that was pure by the criteria of high-performance liquid chromatography and thin-layer chromatography with different solvent systems. The pure product was well separated from all contaminants including one which in high-performance liquid chromatography was not easily separated from N-bromoacetyl-3,3',5-triiodo-L-thyronine. The latter was characterized by 1H nuclear magnetic resonance, plasma desorption mass spectrometry, thin-layer chromatography, high-performance liquid chromatography, UV spectrophotometry, and melting point. Amounts of 3,3',5-triiodo-L-thyronine ranging from picograms, including carrier-free 125I-labeled triiodothyronine, to 200 to 300 mg can be processed with the equipment used in the present investigation.

Comparison of mass spectral techniques using organic peroxides related to artemisinin.

The mass spectra of three peroxides related to artemisinin (1) are compared in nine different ionization modes. Ion trap mass spectrometry (MS/MS) spectra reveal numerous pathways for the electron impact (EI) decompositions. In the EI mode, the best spectra are obtained by using the ion trap mass spectrometer at low temperatures. Loss of oxygen is observed with the other EI spectrometers, suggesting catalytic decomposition in the ion source. Methane positive and negative chemical ionization (CI) spectra show considerable fragmentation, while isobutane CI spectra show only (M + H)+ for 1 and (M + H - H2O)+ for dihydroartemisinin (2) and (3). An unusually abundant (2M + H)+ is observed for 1 in both positive-ion plasma desorption and fast atom bombardment mass spectra.

Californium-252 plasma desorption mass spectrometry of aminoglycoside antibiotics.

This paper presents mass spectral data of eleven aminoglycoside antibiotics by using californium-252 plasma desorption (252Cf PD) mass spectrometry. This mass spectral data could be used to develop a confirmatory method for monitoring aminoglycoside antibiotic residues isolated from food products of animal origin. Mass spectra were determined by applying time-of-flight 252Cf PD mass spectrometry to eleven aminoglycoside antibiotics, namely: neomycin, kanamycin, paromomycin, tobramycin, apramycin, streptomycin, dihydrostreptomycin, amikacin, netilmicin, sisomicin and gentamicins. All eleven antibiotics yielded positive ion spectra. These hydrophilic antibiotics were derivitized to extractable chromopheric compounds. All but two antibiotics (streptomycin and dihydrostreptomycin) yielded nitrophenyl derivatives and spectra were obtained in both negative and positive ion modes. Derivatized aminoglycosides produced cation and anion spectra with quasimolecular ions corresponding to [M + H]+., M+, [M - H]-., [M + Na]+, [M + K]+ and M-. or [M - H]- and M-. or [M - H]-. Underivatized antibiotics were best examined in the positive ion mode. 252Cf PD mass spectrometry consistently produced very strong molecular or quasimolecular ions for all aminoglycoside antibiotics.

Californium-252 plasma desorption mass spectrometry of skin lipids. Positive and negative ions formed by attachment processes.

Californium-252 plasma desorption mass spectra have been run on a series of typical lipids found in skin, including a fatty acid, a methyl and wax ester, a mono-, di- and triglyceride and an anhydride, in a effort to discover the nature of peaks caused by fingerprint contamination of the sample holders. The triglyceride was identified as the source, and peaks found by reattachment of abundant fragment ions to the original lipid were noted in several cases.

Effects of osmotic dehydration on the nucleotides of isolated chromaffin granules: evaluation by 31p nuclear magnetic resonance.

The 31P nuclear magnetic resonance spectrum of chromaffin granules isolated from bovine adrenal medulla, when examined at 30 degrees C in 0.3 M sucrose, shows sharply defined gamma, alpha, and beta nucleotide resonances with linewidths of 141, 58 and 90 Hz respectively. These are essentially unchanged when examined at 4 degrees C. When the granules are resuspended in 1.6 M sucrose, the linewidths of 161, 117 and 162 Hz seen at 30 degrees C reversibly broaden to greater than 1000 Hz at 4 degrees C. Addition of quinacrine produces no discernable change in the nucleotide linewidths of either preparation. The broadening at high osmolarity and low temperature appears to be due to ATP:catecholamine complex formation rather than to increased viscosity. Since purification of chromaffin granules is often performed utilizing sucrose gradients, the present results raise the question of whether dehydration may alter the structure of the granule core.

Synthesis and characterization of methyl 6-O-alpha- and -beta-D-galactopyranosyl-beta-D-galactopyranoside.

Sequential tritylation, acetylation and detritylation of methyl beta-D-galactopyranoside gave crystalline methyl 2,3,4-tri-O-acetyl-beta-D-galactopyranoside (4) and methyl 2,3,6-tri-O-acetyl-beta-D-galactopyranoside, the latter being the minor product resulting from acetyl migration. Reaction of 4 with 2,3,4,6-tetra-O-acetyl-alpha-D-galactosyl bromide in benzene, in the presence of mercuric cyanide and mercuric bromide, gave the alpha- and beta-D-(1----6)-linked disaccharides (7 and 9, respectively) in high yield, and their structure was confirmed by 1H- and 13C-n.m.r. 1d. and 2d. spectroscopy. O-Deacetylation of 7 gave the hitherto unknown, crystalline methyl 6-O-alpha-D-galactopyranosyl-beta-D-galactopyranoside. O-Deacetylation of 9 gave the corresponding, beta-D-linked disaccharide methyl glycoside, the physical constants of which are discussed with respect to controversial data in the literature.

Direct observation of 6-fluorodopamine in guinea pig nerve microsacs by 19F NMR.

19F nuclear magnetic resonance (NMR) has been used to examine the disposition of ring-fluorinated dopamine and norepinephrine in microsacs prepared from striata of guinea pig brains. Following incubation with a 10(-4)M initial concentration of 6-fluorodopamine (6F-DA), intact micmicrosacs at 4 degrees C gave a 19F NMR spectrum in which the 6F-DA present was sufficiently mobile to be visible. Intra-vesicular 6F-DA in striatal nerve terminals thus appears to exist in an environment resembling that in chromaffin vesicles but different from that prevailing inside the amine storage vesicles of platelets. Our data also suggest that the study of fluorinated compounds by 19F NMR can be used to expand our understanding of processes related to amine uptake, metabolism, and storage in nerves.

Isolation and characterization of a unique galactoside from male Drosophila melanogaster.

A ninhydrin-positive compound with presumptive hormonal activity, previously considered to be a peptide (Chen, P.S., and Bühler, R. (1970), J. Insect Physiol. 16, 615), has been isolated from adult male Drosophila melanogaster. Chromatographic analysis of the acid-hydrolyzed material revealed the presence of ethanolamine, phosphorus, galactose, and glycerol. Chemical analysis showed these to be present in equimolar amounts. Based on its phosphorus content, the nonreducing material took up 2 equiv of periodate, and released 1 equiv of formaldehyde. Characterization of the compound as 1-O-(4-O-(2-aminoethyl phosphate)-beta-D-galactopyranosyl)-x-glycerol was achieved by gas chromatography-mass spectroscopy and 1H and 31P NMR using model compounds. In vivo synthesis from labeled precursors is in accord with the proposed structure.

Chemical structure and purity of dyes used in lymphograms.

Because of confusion in the literature, studies were performed on several samples of patent blue violet and Alphazurine 2G to determine their chemical structure and purity. Nuclear magnetic resonance studies revealed that three distinct compounds and one mixture had all been labeled as the same compound. The purity of the dyes varied from batch to batch, as shown by thin-layer chromatography. Caution should be exercised in the use of these dyes for lymphograms.

13C nuclear magnetic resonance spectroscopy of native and recombined lipoproteins.

(13)C nuclear magnetic resonance data on native and recombined lipoproteins are reported. [Methyl-(13)C]phosphatidylcholine, [methyl-(13)C]sphingomyelin, 1,2-[dioleoyl-1-(13)C]-sn-phosphatidylcholine and cholesteryl-[1-(13)C]oleate were enriched with 90% carbon-13 in respective molecules by chemical synthesis and used for recombination experiments with high density lipoprotein apoproteins. Relaxation times for these specifically enriched lipids in organic solvents, (2)H(2)O, and lipid-protein complexes isolated by ultracentrifugal flotation, were measured. The results show that both the phosphatidylcholine and sphingomyelin polar headgroups have the same hydrophilic environment in either sonicated lipid particles or reassembled lipoproteins, and suggest that ionic interaction of lipid and apolipoproteins is of minor importance in the formation of plasma lipoprotein complexes. Our experiments indicate that (13)C nuclear magnetic resonance spectroscopy will contribute to the understanding of lipidprotein interaction in lipoproteins and membranes.

The active site of staphylococcal nuclease: paramagnetic relaxation of bound nucleotide inhibitor nuclei by lanthanide ions.

The structure of 3',5'-thymidine diphosphate bound in the active site of staphylococcal nuclease (EC 3.1.4.7) was studied by measuring the relaxation rate enhancement of substrate analog nuclei by a paramagnetic metal ion. The lanthanide ion, Gd(III), was substituted for Ca(II) in the formation of the ternary complex of nuclease-Gd(III)-3',5'-thymidine diphosphate. Measurements were made of the transverse relaxation rates of protons and the longitudinal and transverse relaxation rates of the phosphorus nuclei of the bound nucleotide. Internuclear distances between the metal ion and atoms of the 3',5'-thymidine diphosphate nucleotide were determined from these data by the Solomon-Bloembergen equation. In general, these distances corresponded closely to those determined by previous x-ray crystallography of the thymidine diphosphate complex.These internuclear distances were also used with a computer program and graphics display to solve for metal-nucleotide geometries, which were consistent with the experimental data. A geometry similar to the structure of the metal-nucleotide complex bound to nuclease determined by x-ray analysis was one of the solutions to this computer modeling process. For staphylococcal nuclease, the nuclear magnetic resonance and x-ray methods yield compatible high resolution information about the structure of the active site. However, differences of uncertain significance exist between the two structures.