The monoterpenes of Artemisia tridentata ssp. vaseyana, Artemisia cana ssp. viscidula and Artemisia tridentata ssp. spiciformis.

Monoterpenes from three different members of the Anthemideae family, Artemisia tridentata ssp. vaseyana, Artemisia cana ssp. viscidula and Artemisia tridentata ssp. spiciformis were isolated and their structures determined using spectroscopic techniques. A total of 26 irregular and regular monoterpenes were identified. Among these, 20 had previously been identified in the Anthemideae family. Of the remaining six, four were known, but previously unidentified in this family. 2,2-Dimethyl-6-isopropenyl-2H-pyran, 2,3-dimethyl-6-isopropyl-4H-pyran and 2-isopropenyl-5-methylhexa-trans-3,5-diene-1-ol were isolated from both A. tridentata ssp. vaseyana and A. cana ssp. viscidula. The irregular monoterpene 2,2-dimethyl-6-isopropenyl-2H-pyran has a carbon skeleton analogous to the biologically important triterpene squalene. Two additional irregular monoterpenes, artemisia triene and trans-chrysanthemal were isolated from A. cana ssp. viscidula and lavandulol was isolated from A. tridentata ssp. spiciformis. This is the first time a compound possessing a lavandulyl-skeletal type has been found in the Anthemideae family.

Thioprenols as hydrazinolysis products of prenylated proteins: dependence upon methylation of the prenylcysteine.

When prenylated proteins are treated with hydrazine at elevated temperatures, a substantial fraction of the prenylcysteines are cleaved at the C-S bond of the beta-carbon of cysteine. Thioprenols, the initial products of this reaction, are then reduced, over time, to hydrocarbons. This elimination reaction is favored several fold if the prenylcysteine is present as a carboxylate derivative rather than as a carboxyl terminal free amino acid. Thus, the pattern of elimination has the potential for detecting substitution (methylation) of prenylcysteines. In addition, the formation of thioprenols leads to more sensitive ways for determination of the prenylcysteines.

Differential prenylation of proteins as a function of mevalonate concentration in CHO cells.

The incorporation of [5-3H]mevalonate into prenylated proteins and polyisoprenoid lipids has been determined as a function of mevalonate concentration in Chinese hamster ovary (CHO) cells that are inhibited in mevalonate synthesis. The relative incorporation of mevalonate into the different end products of isoprenoid metabolism was markedly dependent upon the concentration of mevalonate in the medium. The synthesis of cholesterol was dominant at higher concentrations of mevalonate while higher molecular weight isoprenoids were favored at the lower concentrations. The relative incorporation of mevalonate into the different prenylcysteines of prenylated proteins was dependent upon mevalonate concentration with geranylgeranylcysteine being the principal product at higher concentrations. At low levels of mevalonate farnesylcysteine synthesis predominated and geranylcysteine was detected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from CHO cells that had been radiolabeled at different concentrations of [3H]mevalonate had different patterns on fluorography with relatively few proteins labeled at low concentrations. A study of this effect on the prenylcysteines of a specific protein, Ras, showed considerably less sensitivity to mevalonate concentration than bulk protein. These results indicate that the specific proteins that are prenylated depend upon the availability of the isoprenyl diphosphate substrates.

Quantitation of prenylcysteines by a selective cleavage reaction.

The allylic thioether bond of the prenylcysteines of prenylated proteins has been shown to be cleaved by 2-naphthol under alkaline conditions to yield substituted naphthopyrans. These products are readily resolved from interfering materials by HPLC and have a strongly absorbing chromophore. Thus, this reaction is suitable for quantitative analysis of prenyl substituents of proteins, and we have examined a number of tissues for their content of prenylcysteines. These amino acids are present in mammalian tissues at a concentration of 0.36-1.4 nmol/mg of protein, with a ratio of geranylgeranylcysteine to farnesylcysteine in the range of 4 to 10. Prenylcysteines were also found in the cytosolic fraction of two mouse tissues at about one-third the concentration of the whole organ. The level of these modified amino acids was found to be significantly less in a yeast, a fungus, a brown alga, a higher plant, and an insect. Again, geranylgeranylcysteine is predominant. Prenylcysteines were absent from Escherichia coli but present in an archaebacterium. The prenylcysteine content of mammalian tissue is about 1% of that of cholesterol and about equal to that of ubiquinones and dolichols. Calculations indicate that about 0.5% of all proteins are prenylated.

Prenylated proteins: synthesis of geranylgeranylcysteine and identification of this thioether amino acid as a component of proteins in CHO cells.

Prenylated proteins, labeled in the isoprenoid residue by growing CHO cells in medium containing [5-3H]mevalonate, were degraded by three different proteolytic procedures, enzymatic or alkaline hydrolysis as well as hydrazinolysis. The products thus obtained were analyzed by HPLC with chemically prepared all-trans-geranylgeranylcysteine as a standard. About 10% of the radioactive products released by each lytic procedure showed the same chromatographic properties as geranylgeranylcysteine. This verifies the earlier conclusion, based on less-direct evidence, that this thioether derivative of cysteine is a component of naturally occurring proteins. The finding of this modified amino acid as a product of hydrazinolysis indicates that it is a carboxyl-terminal amino acid and that it is not carboxyl-methylated.

Prenylated proteins: the structure of the isoprenoid group.

The mevalonate-derived portion of a prenylated protein from Chinese hamster ovary cells has been established as diterpenoid (C20). This group is linked to a carboxyl-terminal cysteine as a thioether. It was removed from the protein by hydrazinolysis followed by Raney nickel desulfurization, and the resulting hydrocarbon fraction was analyzed by gas chromatography-mass spectrometry.

Neotropical ant gardens II. Bioassays of seed compounds.

A number of volatile compounds occur on the seeds of taxonomically unrelated ant-garden epiphytes in western Amazonia. In field trials in southeastern Peru, we assayed the responses of ant-garden ants (Camponotus femoratus) to these and structurally similar compounds applied to artificial "seeds" made from zeolite molecular sieves. Benzothiazole,2, present on seeds of eight ant-garden epiphytes, repelled ants over the range of concentrations tested, as did 1-(2-hydroxy-6-methylphenyl)ethanone,3, occurring on seeds of six ant-garden epiphytes. 2-Hydroxy-6-methylbenzoic acid, methyl ester (methyl-6-methylsalicylate, 6-MMS),1, found on seeds of at least nine ant-garden epiphytes, was mildly repellent at high concentration, but stimulated excitement, seed handling, and (rarely) seed carrying at lower concentrations. Vanillin,5, a seed compound of four ant-garden epiphytes, and limonene,6, a monoterpene from seeds of three ant-garden epiphytes, both stimulated excitement, alarm, seed handling, and (rarely) seed carrying. Identified from seeds of seven ant-garden epiphytes, 1-(2,4-dihydroxyphenyl)ethanone,4, elicited little or no response. Among 70 compounds tested (mainly aromatic compounds), those found on seeds of ant-garden epiphytes or having structural features in common with such compounds were the most attractive to the ants. Although not present on epiphyte seeds, 2-hydroxy-3-methoxybenzenemethanol,10, consistently stimulated seed transport to the nest in one year, but did so only rarely in subsequent years. Some of the volatile compounds on seeds of ant-garden epiphytes probably play a role in ant attraction to epiphyte seeds, but evidence remains ambiguous. Finally,Ca. femoratus responded to one test compound [1-(2-hydroxy-4-methoxyphenyl)ethanone,60] (absent from epiphyte seeds) by descending from the vegetation to the ground.

Neotropical ant gardens : I. Chemical constituents.

In ant gardens of lowland Amazonia, parabiotic ant speciesCamponotus femoratus andCrematogaster cf.limata parabiotica cultivate a taxonomically diverse group of epiphytic plants, whose establishment is restricted to arboreal carton ant nests. Epiphyte seeds are collected by workers ofCa. femoratus, the larger of the two ants, and stored unharmed in brood chambers where they subsequently germinate. Although seeds of some ant-garden epiphytes bear nutritional rewards, previous studies have shown that these rewards are not sufficient to explain the pattern of ant attraction to seeds. Five aromatic compounds occur frequently in and on the seeds of most ant-garden epiphytes and may be chemical cues by which ants recognize propagules of their symbiotic plants. The most widely distributed of these is methyl 6-methylsalicylate [6-MMS]1, previously reported as a major mandibular gland product in relatedCamponotus species and present in trace quantities inCa. femoratus males. (-)-Citronellol6 (previously unreported inCamponotus) was the principal volatile constituent in extracts of male heads, and (-)-mellein7 was present in small quantities. Discovery of 6-MMS inside the mandibular glands of maleCa. femoratus (and its presence in analogous glands of related ants) offers preliminary support for Ule's (1906) hypothesis that seeds attract ants by mimicking ant brood. In addition, the likely fungistatic activity of seed compounds suggests that they could retard microbial pathogens of ants and plants in the organic detritus of nest gardens. While the presence of identical seed compounds in so many unrelated plant lineages might represent a remarkable case of convergent evolution, other interpretations are possible.

Prenylated proteins: demonstration of a thioether linkage to cysteine of proteins.

Prenylated amino acid fragments have been isolated from prenylated proteins of Chinese hamster ovary cells. Gel-exclusion chromatography indicates that these proteins are modified by two different prenyl groups. The prenyl-amino acid fragments are labeled by 35S from cysteine, and this bond is cleaved by Raney-Ni, proving that the prenyl residue is linked to protein via a thioether to cysteine. Hydrazinolysis has been used to demonstrate that the cysteine is carboxy terminal.

Dynamics of 6-methoxybenzoxazolinone in winter wheat : Effects of photoperiod and temperature.

6-Methoxybenzoxazolinone (6-MBOA), a compound derivable from some freshly growing plants, is known to stimulate reproduction in some mammals and birds. Winter wheat was studied under controlled laboratory conditions to determine the effects of photoperiod and temperature on derivable 6-MBOA content. Longer photoperiods decrease the amount of derivable 6-MBOA per gram of fresh material in 4-day-old wheat seedlings. Higher temperatures also decrease the amount of derivable 6-MBOA in 4-day-old wheat. 6-MBOA content decreases as the plant ages. Comparisons of only the first centimeter above the seed produced the same age-related result. 6-MBOA is concentrated in the meristematic region with decreasing amounts found in higher portions of the plant. Roots from 9-day-old plants contain 6-MBOA. Unsprouted wheat seeds contain negligible amounts of 6-MBOA. These results demonstrate that environmental variables have a significant effect on derivable 6-MBOA levels, but that under all the regimes studied, 6-MBOA is present in freshly sprouted wheat.