Homobatrachotoxin in the genus Pitohui: chemical defense in birds?

Three passerine species in the genus Pitohui, endemic to the New Guinea subregion, contain the steroidal alkaloid homobatrachotoxin, apparently as a chemical defense. Toxin concentrations varied among species but were always highest in the skin and feathers. Homobatrachotoxin is a member of a class of compounds collectively called batrachotoxins that were previously considered to be restricted to neotropical poison-dart frogs of the genus Phyllobates. The occurrence of homobatrachotoxin in pitohuis suggests that birds and frogs independently evolved this class of alkaloids.

Phenolic antioxidants: potent inhibitors of the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum.

Bis(2-hydroxy-3-tert-butyl-5-methylphenyl)methane (bis-phenol) is the most potent inhibitor of the (Ca2+ + Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum yet identified. The compound behaves as a reversible, tight-binding inhibitor with apparent Ki = 0.3 microM. Butylated hydroxytoluene, butylated hydroxyanisole, and 4-nonylphenol are also effective inhibitors. These observations are of particular interest in light of the widespread use of such phenolic antioxidants and stabilizers in the food industry and in the manufacture of rubbers and plastics and the ease with which the compounds are extracted into organic solvents.

Variability in alkaloid profiles in neotropical poison frogs (Dendrobatidae): genetic versus environmental determinants.

Dendrobatid frogs produce a diverse set of alkaloids, whose profiles appear characteristic of frogs of each species or, in the case of variable species, of each population. In the case of one widespread species, Dendrobates auratus, alkaloid profiles in extracts of skin are markedly different in three populations, one from a Pacific island, Isla Taboga, Panama, one from central mountains in Panama, and the third from the Caribbean coast in Costa Rica. The first contains three major classes of dendrobatid alkaloids, the histrionicotoxins, the pumiliotoxin-A class and the decahydroquinolines. The second contains mainly histrionicotoxins, pumiliotoxin-A class alkaloids and one indolizidine. The third contains histrionicotoxins, a homopumiliotoxin, one decahydroquinoline, and a variety of indolizidines, quinolizidines and pyrrolizidines. Frogs from Isla Taboga or a nearby island were introduced into the Manoa Valley, Oahu, Hawaii, in 1932. Remarkably, although alkaloids of the pumiliotoxin-A class and one decahydroquinoline are still major constituents in skin extracts of Hawaiian frogs descended from the 1932 founding population, histrionicotoxins are absent and a novel tricyclic alkaloid is present. Offspring of wild-caught parents from Hawaii, Panama or Costa Rica raised in indoor terrariums on a diet of crickets and fruit flies do not contain detectable amounts of skin alkaloids. Offspring raised in large outside terrariums in Hawaii and fed mainly wild-caught termites and fruit flies do contain the same profile of alkaloids as their wild-caught parents in Hawaii, but at reduced levels. The genetic, environmental and dietary determinants of alkaloid profiles in dendrobatid frogs remain obscure, in particular the underlying cause for total absence in terrarium-reared frogs.

An uptake system for dietary alkaloids in poison frogs (Dendrobatidae).

The skin of poison frogs (Dendrobatidae) contains a wide variety of alkaloids that presumably serve a defensive role. These alkaloids persist for years in captivity, but are not present in captive-raised frogs. Alkaloids fed to poison frogs (Dendrobates, Phyllobates, Epipedobates) are readily accumulated into skin, where they remain for months. The process can be selective; an ant indolizidine is accumulated, while an ant pyrrolidine is not. Frogs (Colostethus) of the same family, which do not normally contain alkaloids, do not accumulate alkaloids. Such an alkaloid uptake system provides a means of maintaining skin alkaloids and suggests that some if not all such 'dendrobatid alkaloids' may have a dietary origin.

Alkaloids in bufonid toads (melanophryniscus): temporal and geographic determinants for two argentinian species.

Bufonid toads of the genus Melanophryniscus represent one of several lineages of anurans with the ability to sequester alkaloids from dietary arthropods for chemical defense. The alkaloid profile for Melanophryniscus stelzneri from a location in the province of Córdoba, Argentina, changed significantly over a 10-year period, probably indicating changes in availability of alkaloid-containing arthropods. A total of 29 alkaloids were identified in two collections of this population. Eight alkaloids were identified in M. stelzneri from another location in the province of Córdoba. The alkaloid profiles of Melanophryniscus rubriventris collected from four locations in the provinces of Salta and Jujuy, Argentina, contained 44 compounds and differed considerably between locations. Furthermore, alkaloid profiles of M. stelzneri and M. rubriventris strongly differed, probably reflecting differences in the ecosystem and hence in availability of alkaloid-containing arthropods.

Batrachotoxin alkaloids from passerine birds: a second toxic bird genus (Ifrita kowaldi) from New Guinea.

Batrachotoxins, including many congeners not previously described, were detected, and relative amounts were measured by using HPLC-mass spectrometry, in five species of New Guinean birds of the genus Pitohui as well as a species of a second toxic bird genus, Ifrita kowaldi. The alkaloids, identified in feathers and skin, were batrachotoxinin-A cis-crotonate (1), an allylically rearranged 16-acetate (2), which can form from 1 by sigmatropic rearrangement under basic conditions, batrachotoxinin-A and an isomer (3 and 3a, respectively), batrachotoxin (4), batrachotoxinin-A 3'-hydroxypentanoate (5), homobatrachotoxin (6), and mono- and dihydroxylated derivatives of homobatrachotoxin. The highest levels of batrachotoxins were generally present in the contour feathers of belly, breast, or legs in Pitohui dichrous, Pitohui kirhocephalus, and Ifrita kowaldi. Lesser amounts are found in head, back, tail, and wing feathers. Batrachotoxin (4) and homobatrachotoxin (6) were found only in feathers and not in skin. The levels of batrachotoxins varied widely for different populations of Pitohui and Ifrita, a result compatible with the hypothesis that these birds are sequestering toxins from a dietary source.

Beta-alanyl-L-tyrosine. Chemical synthesis, properties and occurrence in larvae of the fleshfly Sarcophaga bullata Parker.

1. A naturally occurring peptide, termed sarcophagine, has been isolated from larvae of the fleshfly Sarcophaga bullata. 2. The physicochemical properties of the natural peptide are identical with those of synthetic beta-alanyl-l-tyrosine. 3. During larval growth the concentration of the dipeptide increases continuously; in the fully grown larva it is the predominant non-protein ninhydrin-positive material. 4. Sarcophagine is rapidly and extensively utilized at the time of hardening and darkening of the insect puparium. 5. By virtue of its much higher solubility than that of free tyrosine, the peptide may serve as a reservoir to provide large amounts of readily available tyrosine for subsequent puparium formation.

A stereospecific synthesis of (+/-)-5,8-disubstituted indolizidines and (+/-)-1,4-disubstituted quinolizidines found in poison frog skins.

An efficient, high-yield stereospecific route to three (+/-)-5, 8-disubstituted indolizidines, (209B (I), 209I (II), 223J (III)) and two (+/-)-1,4-disubstituted quinolizidines (207I (IV), 233A (V)), racemates of alkaloids found in the skins of neotropical and Madagascan poison frogs is reported. The structures of the natural alkaloids were thereby established by chiral GC comparison with the exception of indolizidine 209B (I) for which a natural 209B could no longer be detected.

A new subclass of alkaloids from a dendrobatid poison frog: a structure for deoxypumiliotoxin 251H.

Deoxypumiliotoxin 251H [4], representing a new subclass of the pumiliotoxin-A class of alkaloids, has been isolated from a dendrobatid frog, Epipedobates tricolor. The structure, elucidated by nmr, gc-Ftir, and mass spectral analyses, is proposed to be an 8-methyl-6-(5-hydroxy-2-methylhexylidene)-1-azabicyclo[4.3.0]nonan e. The absolute stereochemistry and the relative configuration of the hydroxyl group are unknown.

Alkaloids from bufonid toads (Melanophryniscus): decahydroquinolines, pumiliotoxins and homopumiliotoxins, indolizidines, pyrrolizidines, and quinolizidines.

Skins of bufonid toads of the genus Melanophryniscus contain several classes of alkaloids: decahydroquinolines, pumiliotoxins, allopumiliotoxins, homopumiliotoxins, both 3,5- and 5,8-disubstituted indolizidines, 3,5-disubstituted pyrrolizidines, and a 1,4-disubstituted quinolizidine. Tricyclic alkaloids, including precoccinelline [193A] and alkaloid 236, an oxime methyl ether, are present in one population of Melanophryniscus stelzneri.

Dietary source for skin alkaloids of poison frogs (Dendrobatidae)?

A wide range of alkaloids, many of which are unknown elsewhere in nature, occur in skin of frogs. Major classes of such alkaloids in dendrobatid frogs are the batrachotoxins, pumiliotoxins, histrionicotoxins, gephyrotoxins, and decahydroquinolines. Such alkaloids are absent in skin of frogs (Dendrobates auratus) raised in Panama on wingless fruit flies in indoor terraria. Raised on leaf-litter arthropods that were collected in a mainland site, such terraria-raised frogs contain tricyclic alkaloids including the beetle alkaloid precoccinelline, 1,4-disubstituted quinolizidines, pyrrolizidine oximes, the millipede alkaloid nitropolyzonamine, a decahydroquinoline, a gephyrotoxin, and histrionicotoxins. The profiles of these alkaloids in the captive-raised frogs are closer to the mainland population ofDendrobates auratus at the leaf-litter site than to the parent population ofDendrobates auratus from a nearby island site. Extracts of a seven-month sampling of leaf-litter insects contained precoccinelline, pyrrolizidine oxime236 (major), and nitropolyzonamine (238). The results indicate a dietary origin for at least some "dendrobatid alkaloids," in particular the pyrrolizidine oximes, the tricyclic coccinellines, and perhaps the histrionicotoxins and gephyrotoxins.

Primary tetradecenyl amines from the ant Monomorium floricola.

In contrast to other ants in the genus Monomorium that produce cyclic amines, extracts of Monomorium floricola contain (Z)-7-tetradecenylamine (1) and (Z)-9-tetradecenylamine (2). The structures of these compounds were established from their spectral data and by comparison with synthetic 2.

Alkaloid 223A: the first trisubstituted indolizidine from dendrobatid frogs.

The structure of alkaloid 223A (1), the first member of a new class of amphibian alkaloids, purified by HPLC from a skin extract of a Panamanian population of the frog Dendrobates pumilio Schmidt (Dendrobatidae) has been established as (5R,6S,8R,9S)- or (5S,6R,8S,9R)-6,8-diethyl-5-propylindolizidine, based on GC-MS, GC-FTIR, and 1H-NMR spectral studies. Three higher homologs of 223A, namely alkaloids 237L (2), 251M (3), and 267J (4), have been detected in other extracts, and tentative structures are proposed.

Investigation of the TCA cycle and the glyoxylate shunt in Escherichia coli BL21 and JM109 using (13)C-NMR/MS.

Acetate accumulation is a common problem observed in aerobic high cell density Escherichia coli cultures. A previous report has hypothesized that the glyoxylate shunt is active in a low acetate producer, E. coli BL21, and inactive in a high acetate producer, JM109. To further investigate this hypothesis, we now develop a model for the incorporation of (13)C from uniformly labeled glucose into key TCA cycle intermediates. The (13)C isotopomer distributions of oxaloacetate and acetyl-CoA are first determined using NMR and MS techniques. These distributions are next validated by predicting the NMR spectrum of glutamate. Under steady state isotopic conditions, and with knowledge of the full isotopomer distributions of oxaloacetate and acetyl-CoA, the flux ratios through the TCA cycle and the glycoxylate shunt are obtained with respect to the flux through the PPC anaplerotic shunt. We conclude that in BL21, the glyoxylate shunt is active at 22% of the flux through the TCA cycle, and is inactive in JM109. Further, in BL21, the flux through the TCA cycle equals the flux through the PPC shunt, while in JM109 the TCA cycle flux is only third of the flux through the PPC shunt.

Ammonia chemical ionization tandem mass spectrometry in structure determination of alkaloids. I. Pyrrolidines, piperidines, decahydroquinolines, pyrrolizidines, indolizidines, quinolizidines and an azabicyclo[5.3.0]decane.

CI-MS/MS of alkaloids with ammonia reagent gas and collision-induced dissociation provides unique structural information for certain mono- and bi-cyclic alkaloids. This technique was applied to solve the structures of 195C, a 4,6-disubstituted quinolizidine alkaloid found in frog skin and an ant, and 275A, a novel 3,5-disubstituted azabicyclo[5.3.0]decane found in frog skin.

A gas chromatographic-mass spectral assay for the quantitative determination of oleamide in biological fluids.

Oleamide is a putative endogenous sleep-inducing lipid which potently enhances currents mediated by GABAA and serotonin receptors. While a quantitative assay would aid in determining the role of oleamide in physiological processes, most of the available assays are lacking in sensitivity. We now describe a quantitative assay for measuring low nanogram amounts of oleamide in biological fluids using GC/MS in the selective ion-monitoring mode. The internal standard (13C18 oleamide) was added to known concentrations of oleamide, which were converted to the N-trimethylsilyl or N-tert-butyldimethylsilyl derivatives before analysis by GC/MS, yielding linear calibration curves over the range of 1-25 ng of oleamide when monitoring the m/z 338/356 fragments. Using this technique, oleamide levels were determined following solvent extraction of normal rat cerebrospinal fluid and plasma to be 44 and 9.9 ng/ml, respectively. This technique constitutes a sensitive and reliable method for determining low nanogram quantities of oleamide in biological fluids.

The chemistry of exploding ants, Camponotus spp. (cylindricus complex).

A detailed comparative analysis of the exocrine chemistry of nine Bruneian Camponotus species in the cylindricus complex is reported. Workers of these species are known to have hypertrophied mandibular glands and release their glandular contents suicidally from the head by rupturing the intersegmental membrane of the gaster. All of the species produce mixtures of polyacetate-derived aromatics, including hydroxyacetophenones, which display pH-dependent color changes, and aliphatic hydrocarbons and alcohols. In addition, three species contained (6R)-2,6-dimethyl-(2E)-octen-1,8-dioic acid (9) or (3S)-8-hydroxycitro-nellic acid (10a), previously unreported from insects. These compounds were characterized from their spectral data, and confirmed by comparison with synthetic samples. The allomonal role of these compounds is based on numerous field observations, and their chemotaxonomic value is presented.

A new class of alkaloids from a dendrobatid poison frog: a structure for alkaloid 251F.

Alkaloids of a new class are present in skin extracts of the dendrobatid poison frog, Minyobates bombetes, of Colombia. The structure of the major alkaloid of this class, 251F, has been determined as a trimethylcyclopenta[b]quinolizidinemethanol 1 by nmr, gc-Ft-ir, and ms studies including ms-ms. At least nine congeners of 251F were detected in these extracts.