Identification of a female-specific, antennally active volatile compound of the currant stem girdler.

We identified (Z)-9-octadecen-4-olide as a female-specific, antennally active compound from the currant stem girdler Janus integer Norton. Female specificity was demonstrated by gas chromatographic comparison of liquid chromatography fractions of male and female volatile emissions and whole body extracts. The gamma-lactone was identified by coupled gas chromatographic-electroantennographic detection (GC-EAD), coupled gas chromatographic-mass spectrometric (GC-MS) analysis, microchemical reactions, and GC and MS comparison with authentic standards. GC-EAD analysis of female volatile emissions and cuticular extracts showed a single peak of activity on male antennae, which was not present in male-derived materials. Female antennae did not respond to any of the tested materials. The hydrogenation product of the natural EAD-active material was a known saturated gamma-lactone. The mass spectrum of the dimethyl disulfide derivative of the natural y -lactone was consistent with a double bond present in the 9 position. Comparison of the natural gamma-lactone and a synthesized racemic mixture of (Z)-9-octadecen-4-olide on a chiral GC column showed the presence of a single enantiomer in the natural material.

Reduction of Carpophilus freemani dobson (Coleoptera: nitidulidae) aggregation pheromone response by synthetic analogues.

Analogues of (2E,4E,6E)-5-ethyl-3-methyl-2,4,6-nonatriene, the major component of the aggregation pheromone of Carpophilus freemani Dobson (Coleoptera: Nitidulidae), were synthesized and the potency of these compounds in suppressing the response of C. freemani to its pheromone in a wind tunnel bioassay was determined. The most potent compounds reduced behavioral response to pheromone 83-96% when the inhibitors were present in 10-fold excess. These compounds are (1Z, 3E,5E)-1-methoxy-3-ethyl-5-methyl-1,3,5-heptatriene, (1E,3E, 5E)-1-cyclopropyl-3-ethyl-5-methyl-1,3,5-heptatriene, and (1Z,3E, 5E)-1-cyclopropyl-3-ethyl-5-methyl-1,3,5-heptatriene. In the presence of fermenting bread dough (a pheromone synergist), the most potent inhibitory compound, (1Z,3E, 5E)-1-cyclopropyl-3-ethyl-5-methyl-1,3,5-heptatriene, was less effective in reducing mean landings (69% vs 99%) than when dough was absent. This inhibitory compound causes a reduction of response to pheromone but does not cause a reduction of response to fermenting food-type volatiles such as fermenting bread dough. Analogues of pheromones that strongly reduce response to pheromones by insects might be useful as biochemical probes to study the pharmacophoric (three-dimensional structure) requirements for pheromone perception.

Physiological responses of rats fed loline and ergot alkaloids from endophyte-infected tall fescue.

Sixty-three male sprague Dawley rats were randomly assigned to dietary treatments containing 1) N-formylloline alkaloid (NFL), 2) N-acetylloline alkaloid (NAL), 3) NFL + NAL, 4) NFL + a mixture of ergot alkaloids, 5) NAL + a mixture of ergot alkaloids, 6) NFL + NAL + a mixture of ergot alkaloids, 7) a mixture of ergot alkaloids, 8) endophyte-free tall fescue seed (EFTF), and 9) endophyte-infected tall fescue (EITF) seed (negative control). All diets were prepared by mixing the prepared treatments and Laboratory Chow (1:1 ratio) and were fed at a maximum of 15 g per rat per day. All rats were killed at termination (d 18). Rats fed the EITF consumed less (P < .05) than those fed all other treatments. Feed intake for rats fed the NFL + ergot alkaloids was lower than for those fed NAL, NAL + ergot alkaloids, NFL + NAL + ergot alkaloids, ergot alkaloids, and EFTF treatments. Average daily weight gains (ADG) followed a trend similar to feed intake with some exceptions. Rats fed the NFL + NAL treatments had higher (P < .05) ADG than those fed all other treatments except the NFL + NAL + ergot alkaloid treatment. Rats fed the EITF had lower (P < .05) ADG than those fed all other treatments except those fed ergot alkaloids and NAL + ergot alkaloids. Epididymides, testes, hypothalamus, corpus striatum weights, prolactin and alkaline phosphatase content were not altered by dietary treatments. Results suggest that loline alkaloids may have a slight depressing effect on feed intake.

Oxidative coupling of mithramycin and hydroquinone catalyzed by copper oxidases and benzoquinone. Implications for the mechanism of action of aureolic acid antibiotics.

The copper oxidases human ceruloplasmin and Polyporous anceps laccase catalyze the oxidative coupling of mithramycin (1) and its aglycone chromomycinone (2) with p-hydroquinone to form new mithramycin-hydroquinone (3) and chromomycinone-hydroquinone adducts (4), respectively. Similar adducts could be formed by the nonenzymatic mimic of this reaction using benzoquinone and these aureolic acids in buffer solutions. FABMS of 3 indicated that the hydroquinone moiety was attached to the aureolic acid aglycone. Acid hydrolysis of 3 yielded a compound with the same chromatographic and spectroscopic characteristics as 4. Structure elucidation of 4 by NMR and MS revealed that the hydroquinone was attached to the C-5 position of the aglycone. NMR evidence indicated that 4 consisted of a mixture of ortho-substituted biphenyl rotamers. The mechanism of the copper oxidase catalyzed adduct formation reaction is presumed to involve radical formation through hydrogen removal at the 8-phenolic position, radical isomerization, and coupling with semiquinone radical also formed during enzymatic and nonenzymatic incubations. Identification of the covalent-hydroquinone adduct provides evidence that aureolic acid antibiotics can be metabolically converted to reactive radical intermediates, and it establishes the C-5 position of aureolic acid as an enzymatically reactive site. Unlike mithramycin, the mithramycin-hydroquinone adducts was inactive in the in vivo P388 leukemic antitumor test system.

Male-produced aggregation pheromone ofCarpophilus obsoletus (Coleoptera: Nitidulidae).

Males ofCarpophilus obsoletus Erichson produce an aggregation pheromone to which both sexes respond. The pheromone was identified by GC-MS as (2E,4E,6E,8E)-3,5,7-trimethyl-2,4,6,8-undecatetraene (1), which is also a minor constituent of the pheromone blends ofC. hemipterus (L.),C. freemani Dobson, andC. lugubris Murray. The pheromone was synergized in wind-tunnel bioassays by propyl acetate, a "host-type" coattractant. In a dose-response study, 50 pg of1, plus propyl acetate, was significantly more attractive than just propyl acetate. Pheromone emission from groups of 65 males, feeding on artificial diet, averaged 2.2 ng/male/day. Emissions from individual males were larger, averaging 72 ng/day and ranging as high as 388 ng/day. Synthetic1 was tested in a date garden in southern California (500 µg/rubber septum), using fermenting whole-wheat bread dough as the coattractant. The pheromone plus dough attracted significantly more beetles than dough alone (means were 4.2 and 0.0 beetles per week per trap). Captured beetles were 54% females. Field trap catches were highest during the months of July and August.

In vitro and in vivo ruminal and physiological responses to endophyte-infected tall fescue.

Relationships between alkaloid compounds in endophyte-infected tall fescue and ruminal metabolism were studied in two experiments. In the first experiment, different combinations of the pyrrolizidine alkaloids, N-formyl and N-acetyl loline, were incubated with ruminal fluid for 0, 24, or 48 h. Rate of disappearance of N-formyl and N-acetyl loline increased over time. After 48 h, disappearance of N-formyl loline and combinations of N-formyl and N-acetyl loline was greater than N-acetyl loline. Significant amounts of N-formyl and N-acetyl loline were metabolized and converted to loline. In the second experiment, abomasally cannulated sheep were fed increasing amounts of endophyte-infected feed to compare diet digestibility, alkaloid metabolism, and physiological responses. Total tract DM digestibility was greatest for the endophyte-free diet, as were ruminal and total tract ADF, ruminal NDF, and total tract CP digestibilities. N-Formyl and N-acetyl loline recoveries averaged 5% from abomasal contents and 0% in feces. Sixty-eight percent of the pyrrolizidine alkaloids recovered in abomasal contents had been metabolizable to loline. Ergot alkaloids administered in the diet were recovered (50 to 60%) in the abomasal contents, but recovery was only 5% in fecal collections. No significant differences occurred in the physiological parameters measured. Results indicate that response to endophyte-infected tall fescue may be influenced by ruminal metabolism.

The effect of alkaloids and seed extracts of endophyte-infected tall fescue on prolactin secretion in an in vitro rat pituitary perfusion system.

The objective of this research was to measure the effects of endophyte-infected tall fescue seed extract and various alkaloids associated with the endophyte on in vitro prolactin secretion by rat hemipituitaries. Rat anterior pituitaries (AP) were dissected into halves and placed in temperature-controlled culture chambers (37 degrees C). The tissue was perfused with culture media at a flow rate of 12 mL/h. After perfusion for at least 90 min with control media, AP halves were exposed to their respective treatments for 15 min before they were returned to the control media. The treatments for Exp. 1 were .01 micrograms of alpha-ergocryptine/mL of culture medium, .01 microgram of ergonovine/mL of culture medium, .01 gram-equivalents of endophyte-infected tall fescue seed/mL of culture medium, and .01 gram-equivalents of endophyte free tall fescue seed/mL of culture medium. Treatments for Exp. 2 consisted of 10(-4), 10(-6), and 10(-8) M concentrations of perloline, N-formyl loline, N-acetyl loline, N-methyl loline, and alpha-ergocryptine. alpha-Ergocryptine suppressed (P less than .10) prolactin secretion in both experiments. Ergonovine and perloline both stimulated (P less than .10) prolactin secretion. The loline alkaloids (N-formyl loline, N-acetyl loline, N-methyl loline) had no effect on prolactin secretion. The endophyte-infected seed extract treatment suppressed (P less than .10) prolactin secretion. The endophyte-free seed extract treatment had no effect on prolactin secretion. In Exp. 2, prolactin secretion from AP responded to alpha-ergocryptine treatment in a dose-dependent fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids of Stipa robusta (sleepygrass) infected with an Acremonium endophyte.

Stipa robusta (= Stipa vaseyi) is a perennial grass found in certain areas of the southwestern United States. It is commonly known as sleepygrass, as horses that ingest this grass may become profoundly somnolent or stuporous for periods of time lasting up to several days. In an attempt to determine the active principle(s), fractionation of a methanolic extract of sleepygrass infected with an Acremonium endophyte has yielded lysergic acid amide (20 micrograms/g dry wt), isolysergic amide (8), 8-hydroxylsergic acid amide (0.3), ergonovine (7), chanoclavine-I (15), and N-formylloline (18). Related alkaloids have been found in many endophyte-infected grasses. The dominant alkaloid constituent in sleepygrass, lysergic acid amide, has not previously been identified in a grass in such high concentration. Lysergic acid amide is likely to be the basis for the extreme sedative effects on animals, given past pharmacological work on the compound from the ergot fungus Claviceps paspali.

Alkaloid toxins in endophyte-infected grasses.

Grasses infected with clavicipitaceous fungi have been associated with a variety of diseases including classical ergotism in humans and animals, fescue foot and summer syndrome in cattle, and rye-grass staggers in sheep. During the last decade it has been recognized that many of these fungal infections are endophytic; a fungal endophyte is a fungus that grows entirely within the host plant. Inspection of field collections and herbarium specimens has revealed that such infections are widespread in grasses. The chemistry associated with these grass-fungal interactions has proved to be interesting and complex, as each grass-fungal pair results in a unique "fingerprint" of various alkaloids, of which some are highly toxic to herbivores. In many cases the presence of an endophyte appears to benefit the plant by increasing drought resistance, or by increasing resistance to attack by insects, thus improving the overall survivability of the grass. This review will focus on alkaloids that have been reported in endophyte-infected grasses.

Measurement of oxygen solubility in fermentation media: a colorimetric method.

Methods of measuring oxygen solubility in culture media are scarce, and those available are tedious to apply. A simple colorimetric assay was developed and applied to the analysis of oxygen solubility during alcoholic fermentation. The method was based on the consumption of oxygen by glucose oxidase activity and the production of the pink quinone of syringaldazine by coupled peroxidase activity. Color formation at 526 nm progressed through an optimum that was a linear function of the oxygen added to the assay. Sensitivity was maximized by operating at pH 7 and limiting the medium sample volume added. Each assay took 10-15 min to prepare and react. Reaction time was minimized by using abundant glucose and enzyme concentrations. Data obtained by the assay developed showed good agreement with published oxygen solubilities in water and selected media at various temperatures. Subsequent analyses of fermentation broths indicated falling sugar concentration to be primarily responsible for increases in oxygen solubility during fermentation. For example, during fermentations started with 230 g/L xylose or glucose, oxygen solubility could increase by 41% due to sugar consumption alone. This procedure can provide the solubility data needed to accurately calibrate in-line electronic probes for monitoring dissolved oxygen concentration during fermentation processes.

Microbial transformations of natural antitumor agents XXII: Conversion of bouvardin to O-desmethylbouvardin and bouvardin catechol.

Bouvardin is a cyclic hexapeptide antitumor agent which undergoes two major microbial transformation reactions. Screening with 220 cultures revealed 17 different strains capable of producing O-desmethylbouvardin in good yield. O-Desmethylbouvardin was isolated and characterized from preparative scale incubations with Streptomyces rutgersensis NRRL B-1256. Four aspergilli and one streptomycete formed bouvardin catechol when O-desmethylbouvardin was used as substrate. Bouvardin catechol was isolated and characterized from a preparative scale incubation with Aspergillus ochraceous UI 398.

Analysis, Production, and Isolation of an Extracellular Laccase from Polyporus anceps.

Methods are described for the analysis, production, and isolation of laccase produced by a strain of Polyporus anceps. A simple quantitative colorimetric assay based on the oxidation of syringaldazine to syringaldazine quinone is described. Using a defined medium supplemented with the amino acids cysteine and histidine and with elevated phosphate, consistently high titers of laccase were obtained. The enzyme was isolated directly from fermentation medium by binding to diethylaminoethyl cellulose, and, once bound to the ion exchanger, it could be stored for 6 months at -70 degrees C with minimal loss of activity. The enzyme was quantitatively recovered from the resin by elution with 0.2 M phosphate buffer (pH 5.0).

Effect of chemotactic factors on calcium levels of rabbit neutrophils.

Studies were undertaken to provide a systematic investigation of the effect of the chemotactic factor, formyl-methionyl-leucyl-phenylalanine (F-Met-Leu-Phe), on the levels of total and exchangeable calcium in rabbit neutrophils. The 45Ca2+ specific activity of the cells was thus determined before and after stimulation by chemotactic factors. Total cell calcium was found to be constant in neutrophils equilibrated with 50-1,000 microM extracellular calcium but decreased by 30% at an extracellular calcium concentration of 5 microM. F-Met-Leu-Phe had little, if any, statistically significant effect on the level of total cell calcium. In contrast, F-Met-Leu-Phe affects greatly the steady-state levels of radioactive calcium in the neutrophils. The primary effect is an initial loss followed by an increase of cell-associated radiolabeled calcium. The extent and even the direction of the effect depends on the level of extracellular calcium as well as the concentration of the chemotactic factor and the duration of its interaction with the cell. The results are discussed in terms of the hypothesis that the binding of the chemotactic factor to its receptor leads to, among other things, a graded displacement of previously bound Ca2+.