Cembrene A and a congeneric ketone isolated from the paracloacal glands of the Chinese alligator (Alligator sinensis).

A diterpene hydrocarbon and diterpene ketone isolated from the paracloacal gland secretions of the Chinese alligator (alligator sinensis) were examined by GC-MS and 1H-NMR, 13C-NMR, and IR spectroscopy, and identified as 4,8,12-trimethyl-1-(1-methylethenyl)-3,7,11-cyclotetradecatrien e (cembrene A, 1) and its congeneric ketone, 4,8,12-trimethyl-1-(1-methylethenyl)-3,7-cyclotetradecadien- 10-one (2), respectively. This is the first report of cembrene A from a vertebrate; the ketone has not been described previously.

9(10)-alkoxystearic acids lengthen the circadian period of a fatty acid auxotroph of Neurospora crassa.

9- and 10-Alkoxystearic acids with alkoxy groups of varying size were synthesized by solvomercuration of methyl oleate. These "knobbed" fatty acids were used to supplement a Neurospora strain carrying the cel mutation, which confers a partial fatty acid deficiency. Supplemental unsaturated or short-chain fatty acids are known to lengthen dramatically the circadian period of spore formation (conidiation) in cel strains. A similar dramatic lengthening was obtained with the alkoxy supplements. The change in period, however, did not correlate positively with the size of the knob. Smaller knobbed analogs gave somewhat greater period lengthening and were removed from the medium faster. It may be that the knobbed analogs cause period lengthening by an indirect effect on Neurospora membranes rather than, or in addition to, a direct perturbation.

Preparation of functional group analogs of unsaturated fatty acids and their effects on the circadian rhythm of a fatty-acid-deficient mutant of Neurospora crassa.

Functional group analogs of oleic, linoleic and linolenic acids were prepared by coverting their double bonds to dibromo, cyclopropyl, epoxy, methoxy, and, in the case of oleic acid, hydroxy groups. These compounds were supplemented to the bd csp cel strain of the mold Neurospora crassa. The cel mutation confers a partial requirement for saturated fatty acids and, also, perturbs the circadian rhythm of spore formation. For example, the period of bd csp cel's rhythm is dramatically lengthened upon supplementation by natural cis-unsaturated fatty acids. Of the analogs tested, only the monoepoxy, monomethoxy, dibromo, and hexabromo stearic acids gave significant period lengthening. Other analogs, which should have comparable abilities to disrupt lipid bilayer packing, gave no rhythm effect. Further, the inactive di- and tri-methoxystearic acid analogs were incorporated to a greater extent than the active mono-methoxystearic acid. The results do not, therefore, support a direct alteration in membrane "fluidity' as the mode of action of the period-lengthening fatty acids.

Unsaturated fatty acid isomers: effects on the circadian rhythm of a fatty-acid-deficient Neurospora crassa mutant.

The fatty acids oleic, linoleic, and linolenic, each of which has a cis double bond at the delta 9 position, are known to lengthen the circadian period of conidiation (spore formation) of strains of Neurospora crassa carrying the cel mutation. cel confers a partial fatty acid requirement on the organism and has been used to promote incorporation of exogenous fatty acids. To test whether a physical effect imparted by the cis double bonds, such as increased membrane fluidity, is critical for the perturbation of the rhythm, various isomers of these fatty acids were supplemented to the bd csp cel strain. Positional isomers of oleic acid, such as petroselinic (delta 6) and vaccenic (delta 11) acids, and longer-chain isomers, such as eicosenoic (delta 11) and erucic (delta 13) acids, did not lengthen the rhythm. The shorter-chain palmitoleic (delta 9) acid did not give a consistent lengthening of the rhythm; it may be elongated to vaccenic acid. In contrast, gamma-linolenic acid (delta 6,9,12) dramatically lengthened the period. Linoelaidic acid (the trans,trans isomer of linoleic acid) lengthened the period at 22 degrees C, but elaidic acid (the trans isomer of oleic acid) did not. Elaidic acid was shown to exert a lengthening effect, but only at lower temperatures. The data do not support a direct physical action as the source of the fatty acids' "chronobiotic" ability.

Circadian rhythms in Neurospora crassa: a mutation affecting temperature compensation.

The circadian rhythm of conidiation (spore formation) in Neurospora crassa is known to be temperature compensated, that is, the period is only slightly affected by the incubation temperature. Thus, the Q10 (the relative rate enhancement corresponding to a 10 degrees C rise in temperature) of the rhythm of the bd csp strain from 14 to 30 degrees C was 1.1, whereas the Q10 of the uncompensated growth rate in the same interval was 2.4. A mutation at the cel locus resulted in loss of the temperature-compensation property in cultures grown below 22 degrees C. The Q10 of the rhythm below 22 degrees C was 2.2, and periods of about 40 hr were observed. In contrast, the Q10 of the rhythm above 22 degrees C was 1.1, with circadian periods of 18-21 hr. Thus, cel displayed a threshold temperature or "break point" for the temperature compensation of its rhythm. Supplementation of cel strains, which require fatty acids, with unsaturated or short-chain fatty acids raised the threshold temperature to 26 degrees C, whereas supplementation with long-chain saturated fatty acids lowered it to 18 degrees C. These data suggest a role for fatty acids, as liquid components or as cellular metabolites, in the mechanism of temperature compensation.