Purification, identification, concentration and bioactivity of (Z)-7-dodecen-1-yl acetate: sex pheromone of the female Asian elephant, Elephas maximus.

In their natural ecosystems, adult male and female Asian elephants, Elephas maximus, live separately. For several weeks prior to ovulation, female elephants release a substance in their urine which elicits a high frequency of non-habituating chemosensory responses, especially flehmen responses, from male elephants. These responses occur prior to, and are an integral part of, mating. Using bioassay-guided fractionation, quantitatively dependent on these chemosensory responses, a specific sex pheromone was isolated and purified by an alternating series of organic and/or aqueous extractions, column chromatography, gas chromatography and high-performance liquid chromatography. Using primarily 1H-proton nuclear magnetic resonance (NMR) spectrometry and gas chromatography-mass spectrometry (GC-MS) of the urine-derived pheromone and its dimethyl disulfide derivative, we determined the structure of the active compound to be (Z)-7-dodecen-1-yl acetate (Z7-12:Ac). Concentrations of Z7-12:Ac in the female urine increased from non-detectable during the luteal phase to 0.48 microgram/ml (0.002 mM) early in the follicular phase and to 33.0 micrograms/ml (0.146 mM) just prior to ovulation. Bioassays with commercially available authentic synthetic Z7-12:Ac, using 10 Asian male elephants at several locations in the US, demonstrated quantitatively elevated chemosensory responses that were robust during successive tests, and several mating-associated behaviors. Bioassays with Z7-12:Ac with adult male elephants dwelling in more natural social situations in forest camps in Myanmar revealed some differing contextual pre-mating behavioral components. The remarkable convergent evolution of this compound suggests that compounds identified in mammalian exudates that are also present in pheromone blends of insects should be re-evaluated as potential mammalian chemosignals.

Female-to-male sex pheromones of low volatility in the Asian elephant,Elephas maximus.

In their natural ecosystems, the sexes of Asian elephants,Elephas maximus, live separately. For several weeks prior to ovulation, the urine and cervical mucus of female Asian elephants contain extractable chemical agents of low volatility that elicit a high frequency of flehmen responses from bull elephants as an integral part of mating. Subsequent to flehmen responses, male sexual arousal occurs and, if the female is available, mating results. During the course of our project to determine the agent(s) and describe the responses associated with female to male sexual communication, we have identified an unusual compound. This compound, apparently the sole component of the active fraction, was identified by mass, proton nuclear magnetic resonance, ultraviolet/visible, and infrared spectrometries as indolo-[2,1-b] quinazoline-6,12-dione (tryptanthrine). Exhaustive and repetitive bioassays established that pure authentic (synthetic) tryptanthrine was not the compound responsible for the bioresponse. Rather a coeluting minor component, also of low volatility, elicited the male bioresponse.

Significant conformational changes in an antigenic carbohydrate epitope upon binding to a monoclonal antibody.

Transferred nuclear Overhauser enhancement spectroscopy (TRNOE) was used to observe changes in a ligand's conformation upon binding to its specific antibody. The ligands studied were methyl O-beta-D-galactopyranosyl(1----6)-4-deoxy-4-fluoro-beta-D-galactopyra nos ide (me4FGal2) and its selectively deuteriated analogue, methyl O-beta-D-galactopyranosyl(1----6)-4-deoxy-2-deuterio-4-fluoro-beta -D- galactopyranoside (me4F2dGal2). The monoclonal antibody was mouse IgA X24. The solution conformation of the free ligand me4F2dGal2 was inferred from measurements of vicinal 1H-1H coupling constants, long-range 1H-13C coupling constants, and NOE cross-peak intensities. For free ligand, both galactosyl residues adopt a regular chair conformation, but the NMR spectra are incompatible with a single unique conformation of the glycosidic linkage. Analysis of 1H-1H and 1H-13C constants indicates that the major conformer has an extended conformation: phi = -120 degrees; psi = 180 degrees; and omega = 75 degrees. TRNOE measurements on me4FGal2 and me4F2dGal2 in the presence of the specific antibody indicate that the pyranose ring pucker of each galactose ring remains unchanged, but rotations about the glycosidic linkage occur upon binding to X24. Computer calculations indicate that there are two sets of torsion angles that satisfy the observed NMR constraints, namely, phi = -152 +/- 9 degrees; psi = -128 +/- 7 degrees; and omega = -158 +/- 6 degrees; and a conformer with phi = -53 +/- 6 degrees; psi = 154 +/- 10 degrees; and omega = -173 +/- 6 degrees. Neither conformation is similar to any of the observed conformations of the free disaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of alkaline borohydride treatment on N-linked carbohydrates of glycoproteins.

The effects of treatments of the glycoprotein ribonuclease-B, the proteins ribonuclease-A and myoglobin, and the glyco-amino acid GlcNAc beta(1-N)Asn with alkali, alkaline sodium borohydride, and aqueous sodium borohydride were systematically studied as a function of the concentration of the reagents, the temperature, and the length of the treatment. High-field 1H-NMR spectroscopy, chromatographic methods and amino-acid analysis were used to characterize products of the treatments of the various compounds. Our results indicate that mild alkaline borohydride treatment, as well as aqueous borohydride treatment alone, is capable of extensively degrading polypeptides and of partially releasing the N-linked glycans from ribonuclease-B. Initially, glycopeptides are produced, the peptide portion of which consists of several amino acids, which are further hydrolyzed to yield a mixture of glyco-asparagines and oligosaccharide-alditols in the ratio of approximately 4:1. Strong alkaline borohydride treatment of ribonuclease-B is capable of completely releasing the N-linked carbohydrates as oligosaccharide-alditols.

Stable glucopyranosylpalladium complexes with cis-beta-hydrogen. A six-membered ring metallocycle with an oxygen donor ligand.

Two stable glucopyranosylpalladium complexes, chloro[1,3-dimethyl-5-(3,4,6-tri-O-acetyl-2-deoxy-alpha-D -arabinohexopyranosyl)-2,4(1H,3H)-pyrimidinedionnato] (triphenylphosphine)-palladium and the corresponding triphenylarsine analog, were studied using fast atom bombardment mass spectrometry, 1H, 13C and 31P nuclear magnetic resonance, UV and IR spectroscopy to establish structures for these complexes. The data obtained indicate that the pyranosyl ring is in a chair conformation in which palladium (C2'), acetoxy (C3' C4') and acetoxymethyl (C5') are equatorial and 1,3-dimethyl-2,4(1H,3H) pyrimidinedion-5-yl (C1') is axial. The palladium(II) ion is encompassed in a six-membered ring metallocycle in which C2' of the glucopyranosyl ring and the oxygen of the C4 carbonyl of the pyrimidinedionyl group occupy adjacent ligand sites. The other two ligand sites on square planar palladium are occupied by triphenylphosphine (or triphenylarsine) cis to C2' and trans to carbonyl oxygen, and chloride trans to C2' and cis to oxygen. This stable metallocycle has three unusual features, a cis-beta-hydrogen, a six-membered Pd-containing ring and an oxygen donor ligand. Its surprising stability is due to conformational barriers to the proper alignment of Pd with pyranosyl ring substituents required for elimination reactions.

Sex pheromone of a conifer-feeding budworm,Choristoneura retiniana, Walsingham.

Sex pheromone components collected from femaleChristoneura retiniana included 11-tetradecenyl acetates and alcohols. The major component wasE-11-tetradecenyl acetate (E11-14∶Ac) with a lesser amount ofZ isomer necessary to induce male response. A 92∶8 ratio ofE,Z11-14∶ Ac appeared optimal. The alcohol component was present at about 10% of the total pheromone mixture, and traps baited with acetates plus alcohol surpassed unmated females in their degree of attractiveness. Chemical analysis indicated a 9∶:1 ratio of theE - Z isomers of 11-tetradecenyl alcohol (11-14∶ol) pheromone components, although bait formulations containing a predominance of either theE orZ isomers were equally successful in field bioassays. Based on male response to traps,E- andZ11-14∶Ac (92∶8E - Z) are essential pheromone components for long-range sex attraction. The 11-14∶ ol enhanced attraction when added at 10% of the total pheromone blend.

Asian bull elephants: Flehmen-like responses to extractable components in female elephant estrous urine.

Flehmen-like responses (urine tests) are one of the characteristic behavioral reactions of male Asian elephants (Elephants maximus) to cow elephants in estrus. Components of the urine of estrous cow elephants were extracted with organic solvents and partially purified by chromatography and shown to evoke Flehmen-like responses when they were presented to adult bulls.

Buckthorn neuropathy in vitro : evidence for a primary neuronal effect.

The neuropathy produced by toxins isolated from the buckthorn shrub (Karwinskia humboldtiana) has previously been proposed as a model for primary PNS demyelination in vivo. These toxins have been suggested to act through a metabolic effect on the Schwann cell. The aim of the present study was to use myelinated organotypic cultures of nervous tissue to dissect further the pathologic effects of these compounds. Combination cultures of mouse spinal cord and dorsal root ganglia were exposed to buckthorn toxins T/496 or T/544 at doses of 1-5 microgram/ml for periods of 48 hours (h) to 14 days. Ultrastructural findings from toxin-treated cultures indicated a primary effect upon the PNS axon, both myelinated and unmyelinated. The effect was expressed mainly as a widening of the periaxonal space, and also as a redistribution of most axonal organelles to a marginal position within the axon or to their close association with stacks of smooth endoplasmic reticulum, leaving more central regions occupied largely by neurofilaments. These changes were followed by focal axonal swelling, floccular disruption of the axoplasm and, ultimately, Wallerian degeneration. Myelin degeneration and chromatolytic changes in ganglion cell bodies appeared to be secondary to axonal alterations. This tissue culture model reproduces many of the features of buckthorn intoxication in vivo. However, the present results suggest that the toxic effect is primarily upon the axon rather than the Schwann cell, and would appear to be consistent with an abnormality in axoplasmic transport.

Chemistry and field evaluation of the sex pheromone of western spruce budworm,Choristoneura occidentalis, Freeman.

Chemical analyses and field bioassays showed a mixture of 92% (E)- and 8% (Z)-11-tetradecenal to be the sex attractant pheromone of the western spruce budworm,Choristoneura occidentalis Freeman. Females were also found to emit small amounts of the corresponding acetates and alcohols, but these components were not active in the bioassay. In contrast, in whole female tip extracts, (E)-11-tetradecenyl acetate predominates, and aldehyde pheromone components are present in lesser quantities, suggesting that final biosynthesis of pheromone takes place just prior to or during emission. At release rates approximating that of the female (2-4 ng/hr), a 92∶8E∶Z blend of the synthetic aldehydes was at least as attractive as live females. Addition of the corresponding acetates or alcohols up to 50% of the aldehyde content did not significantly enhance or inhibit attraction. No major differences were apparent in pheromone production of females from a laboratory stock or from field collections from diverse geographic locations ranging from Colorado to British Columbia.

Sterols of human gallstones: the recent identification of eight different digitonin precipitable sterols.

In this study, we report for the first time the presence of many noncholesterol sterols in human gallstones. Gallstones were obtained from 17 patients at cholesystectomy. They were rich in sterol composition, some 52.8% by weight. A number of different sterols were found: 22-dehydrocholesterol 0.29% of total sterols; cholestanol, 1.54%; lathosterol, 1.92%; 24-methylene cholesterol and campesterol, 0.64%; stigmasterol, 0.02%; beta-sitosterol, 0.21%; and cholesterol, 95.33%. These individual sterols were identified by a variety of methods which included final verification by combined gas chromatography-mass spectroscopy. These unusual sterols were derived either from the diet or from the metabolism of cholesterol in the body. Their presence might well affect the lithogenicity of the bile and raises questions about their metabolism and function generally in the body.

Polyamine metabolites and conjugates in man and higher animals: a review of the literature.

The current state of knowledge of the metabolic fate of the di- and polyamines putrescine, spermidine, and spermine is reviewed. Acetylated, oxidized, and protein-bound polyamines are found to be widely distributed in man and animals. The importance of understanding the roles of these compounds in health and disease is now recognized and is currently the subject of active research.

Polyamine-pyridoxal Schiff bases in urine.

Schiff bases of the diamines 1,3-diaminopropane, putrescine, and cadaverine and the polyamines spermidine and spermine with pyridoxal or pyridoxal phosphate occur in human urine, as shown by gas chromatographic/mass spectrometric selected ion-monitoring techniques. By use of synthetic standards, procedures were devised for conversion of the Schiff bases to stable derivatives amenable to gas chromatographic/mass spectrometric analysis. These procedures involve borohydride reduction of the C = N double bond, hydrolytic removal of the phosphate group, chromatographic separation from the bulk of urinary constituents, and trifluoroacetylation of polar functional groups. The levels of the polyamine-pyridoxal Schiff bases were estimated to be in the range of pmol/ml or urine.

Human transfer factors: structural properties suggested by HPRP chromatography and enzymatic sensitivities.

Leukocyte extracts containing human transfer factor (TF) were fractionated by exclusion chromatography, and the active fraction (Sephadex G25, Fraction IIIa) was subjected to high pressure, reverse phase (HPRP) chromatography and enzymatic degradation. TF activity was assessed by the systemic transfer of dermal skin test reactivity from KLH-immunized donors to naive recipients. Preparative HPRP chromatography resolved Fraction IIIa into multiple chromophoric regions, two of which demonstrated transfer of KLH reactivity. Alkaline phosphatase treatment of Fraction IIIa converted the major ultraviolet-absorbing component, 5'-inosine monophosphate, to inosine and resulted in TF activity being restricted to one region. This HPRP region (R1A) contained less than 1% of the UV254 active material in Fraction IIIa but greater than 90% of the reactivity. The sensitivity of TF to pronase, proteinase K, phosphodiesterase I, and phosphodiesterase II was evaluated by inhibition of systemic transfer of KLH reactivity. Pronase and proteinase K destroyed systemic transfer activity and the pronase destruction could be inhibited with traysylol. Phosphodiesterase I, a 3' exonuclease, destroyed activity, whereas phosphodiesterase II, a 5' exonuclease, did not. The data are consistent with a phosphodiester-containing polypeptide in the structure of human TF for KLH reactivity.

Determination of Indole-3-acetic Acid in Douglas Fir Using a Deuterated Analog and Selected Ion Monitoring: Comparison of Microquantities in Seedling and Adult Tree.

Indole-3-acetic acid (IAA) content in shoot tips of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) trees and seedlings was determined by combined gas chromatography-mass spectrometry using a deuterated analog (d(2)-IAA) as an internal standard and the technique of selected ion monitoring. Ratios of the peak heights of the deuterated analog internal standard to endogenous IAA revealed a slightly higher content of IAA in seedlings compared with the shoot tips collected in June. The relatively high level of IAA (2.9 micrograms per gram fresh weight) in seedlings is discussed in relation to in vitro propagation of this species.

1-N-Acetylspermidine: occurrence in normal human serum.

Using gas chromatography mass spectrometry following derivatization (N-trifluoroacetylation), 1-N-acetylspermidine was found to occur in normal human serum at a level of approximately 0.008--0.5 nmol ml(-1), i.e. about two orders of magnitude lower than the corresponding spermidine level. No evidence for the occurrence in serum of the isomeric 8-N-acetylspermidine was found. Mass spectra of authentic samples of trifluoroactylated 1-N- and 8-N-acetylspermidine are presented.