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1. The plasma concentration, main route of metabolism and excretion of 3H-L-659,989 were studied in male and female rhesus monkeys by dosing either i.v. or orally at 10 mg/kg. 2. The percentage of the AUC for the plasma radioactivity concentration-time curve of oral vs i.v. dosed monkeys was 78% for males and 90% for females, indicating that the dose was well absorbed. 3. The bioavailability of the drug was low (less than or equal to 10%) for all monkeys, probably due to rapid first pass metabolism. The drug was metabolized-predominantly at the C-4'-propoxy side-chain. The two major plasma metabolites were identified as the 4'-2-(hydroxy)propoxy metabolite (3H-trans-4'-HP) and the 4'-hydroxy metabolite (3H-4'-hydroxy) which was isolated as a 2:1 mixture of (+/-)trans: (+/-)cis. 4. Approx. 80% of the radiolabelled dose was excreted equally in the urine and faeces in 96 h, with the largest percentage of the tritiated dose (31 +/- 4%) in the 0-24 h urine. 5. The major metabolites in the excreta were the (+/-)trans/(+/-)cis mixture of 3H-4'-hydroxy and the glucuronide conjugate of 3H-trans-4'-hydroxy. The glucuronide conjugate of 3H-trans-4'-hydroxy was excreted in the urine of i.v. and orally dosed monkeys and represented an average of 21% and 5.1% of the dose, respectively. 3H-4'-Hydroxy was excreted in both the urine and faeces, accounting for less than or equal to 0.1% and 7.4% of the dose in i.v. and orally dosed monkeys, respectively.

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The metabolism of the PAF antagonists kadsurenone and tritium-labeled 9,10-dihydrokadsurenone was studied in rhesus monkeys and rat liver microsomes. The monkey metabolites of the two drugs were isolated as their glucuronide conjugates from the urine of iv dosed males. The metabolites from both monkey and microsomal metabolism were purified by reverse phase HPLC and identified by spectral (NMR, UV, and mass spectrometric) analysis. The principal pathway of biotransformation of the tritium-labeled 9,10-dihydrokadsurenone in monkeys was hydroxylation of the C-5 propyl side chain to give two metabolites, 10-hydroxy-9,10-dihydrokadsurenone and 9-hydroxy-9,10-dihydrokadsurenone. These compounds were excreted as glucuronides. Microsomal incubation of tritium-labeled 9,10-dihydrokadsurenone yielded the 10-, 9-, and 8-hydroxy-9,10-dihydrokadsurenone as major metabolites. Kadsurenone was also metabolized at the C-5 side chain, an allyl group. The monoglucuronide of 9,10-dihydroxykadsurenone was isolated from monkey urine. Spectral analysis was not definitive as to the site of conjugation, and the structure of the metabolite was assigned as the C-10 conjugate. A major metabolite of rat liver microsomal incubation of kadsurenone was 9,10-dihydroxykadsurenone.

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L-659,066 has been characterized as a potent and selective alpha-2 adrenoceptor antagonist. Both in vitro and in vivo, L-659,066 exhibited specificity (comparable to rauwolscine) for alpha-2 over alpha-1 adrenoceptors. Studies comparing L-659,066 with a previously described antagonist, L-657,743, demonstrate that the new compound penetrates the blood-brain barrier only poorly after systemic administration. With a pA2 of 8.44 at alpha-2 adrenoceptors in the isolated rat vas deferens and an IC50 of 3.0 nM against the binding of [3H]rauwolscine to rat cerebrocortical membranes, L-659,066 possessed, respectively, about one-eighth and one-third of the potency of L-657,743. Similar relative potencies were obtained in vivo in pithed rats with regard to blocking peripherally located postjunctional and prejunctional alpha-2 adrenoceptors (L-659,066 = one-seventh and one-fourth of L-657,743, respectively). In tests carried out in vivo with rats for ascertaining alpha-2 adrenoceptor antagonism in the central nervous system--namely, accumulation of cortical dopa and antagonism of mydriasis induced by the alpha-2 agonist, clonidine--L-659,066 had, respectively, less than 1/345th and about 1/5000th of the potency of L-657,743. In mice, L-659,066 had, respectively, approximately 1/29th and 1/1400th of the potency of L-657,743 as an antagonist in vivo of the predominately peripherally mediated inhibition of colonic propulsion caused by clonidine as compared with the mainly centrally mediated antinocisponsive action elicited by the alpha-2 agonist UK 14,304. The foregoing findings are consistent with poor penetration of the blood-brain barrier by L-659,066.(ABSTRACT TRUNCATED AT 250 WORDS)

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In order to discover the intracellular location of luciferase in dinoflagellates, sections from a number of species were treated with a polyclonal anti-luciferase and the bound antibody was visualized at the electron-microscope level by indirect immunogold labelling. In two species of Pyrocystis and in Noctiluca, as in Gonyaulax, antibody became bound to dense vesicles, which correspond in size and position to light-emitting bodies detected in previous work. These vesicles resemble microsomes, are bounded by a single membrane and sometimes project into the vacuole. Unexpectedly, the trichocysts of Gonyaulax and Noctiluca and the related mucocysts of Pyrocystis also bound the antibody. This cross-reaction seems quite independent of bioluminescence, since the trichocysts of the non-luminous Cachonina also reacted positively. The possibility is discussed that a protein, different from luciferase but having some antigenic similarity, is present in trichocysts and related organelles.

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Cultures of Gonyaulax polyedra Stein maintained in the laboratory for 15 to 20 years, including an axenic strain isolated in 1960, have gradually lost the ability to survive in darkness. G. polyedra (70A), isolated in 1970 and maintained in a 12:12 light:dark cycle, now tolerates continuous darkness for a much shorter time than a strain isolated in 1981. I have compared the properties of strain 70A with those of this newer strain (81N), to investigate changes in Gonyaulax with length of time in culture, which may account for poor survival in darkness. When grown in continuous light (13, 12, or 4.5 watts per square meter), strains 70A and 81N have similar growth rates, yields, cell diameters, protein contents, C/N ratios, respiration rates, pigment complements, and photosynthetic rates. When entrained by a light:dark cycle (12L:12D), 70A showed no photosynthesis rhythm, although such a rhythm was formerly present. However, the circadian rhythms in bioluminescence and cell division were normal in both strains. Thus, the circadian clock is apparently still intact in 70A as in 81N. The rate of photosynthesis in strain 70A was constant at a low level, the consequent smaller accumulation of photosynthetic products probably accounting for the limited survival in darkness. The defect in strain 70A may be the loss of a component either directly affecting P(max) or necessary for transduction from the circadian clock to photosynthesis.

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The circadian rhythms in bioluminescence and photosynthesis in Gonyaulax polyedra suspended in unsupplemented sea water have been compared to the same rhythms in f/2, an enriched seawater medium. Cells suspended in sea water for 2 days in continuous light (450 microwatts per square centimeter) showed significantly shorter circadian periods and lower amplitudes than did cells in f/2 medium (a period of 22.2 hours as compared to 23.5 hours). Both period and amplitude changes could be completely reversed by the addition of nitrate at one-fourth or more of the concentration in f/2 medium (0.88 millimolar). The addition to autoclaved seawater of phosphate, vitamins, minerals, or soil extract in concentrations present in f/2 medium had no effect. Thus, the shortening of the circadian period is the consequence of reduced nitrogen supply. Since both the rhythms in bioluminescence and photosynthesis showed similarly shortened circadian periods and lower amplitudes, it is probable that the depletion of nitrate directly affects the circadian clock.

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Cell-free extracts that show activity in photosynthetic electron flow have been prepared from the unicellular dinoflagellate, Gonyaulax polyedra. Electron flow, as O(2) uptake, was measured through both photo-system I and II from water to methyl viologen, through photosystem I alone from reduced 2,6-dichlorophenol indophenol to methyl viologen which does not include the plastoquinone pool or from duroquinol to methyl viologen which includes the plastoquinone pool. Electron flow principally through photosystem II was measured from water to diaminodurene and ferricyanide, as O(2) evolution. Cultures of Gonyaulax were grown on a 12-hour light:12 hour dark cycle to late log phase, then transferred to constant light at the beginning of a light period. After 3 days, measurements of electron flow were made at the maximum and minimum of the photosynthetic rhythm, as determined from measurements of the rhythm of bioluminescence. Photosynthesis was also measured in whole cells, either as (14)C fixation or O(2) evolution. Electron flow through both photosystems and through photosystem II alone were clearly rhythmic, while electron flow through photosystem I, including or excluding the plastoquinone pool, was constant with time in the circadian cycle. Thus, only changes in photosystem II account for the photosynthesis rhythm in Gonyaulax.

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Dividing pairs or single cells of the large dinoflagellate, Pyrocystis fusiformis Murray, were isolated in capillary tubes and their morphology was observed over a number of days, either in a light-dark cycle or in constant darkness. Morphological stages were correlated with the first growth stage, G(1), DNA synthesis, S, the second growth stage, G(2), mitosis, M, and cytokinesis, C, segments of the cell division cycle. The S phase was identified by measuring the nuclear DNA content of cells of different morphologies by the fluorescence of 4', 6-diamidino-2-phenylindole dichloride.Cells changed from one morphological stage to the next only during the night phase of the circadian cycle, both under light-dark conditions and in continuous darkness. Cells in all segments of the cell division cycle displayed a circadian rhythm in bioluminescence. These findings are incompatible with a mechanism for circadian oscillations that invokes cycling in G(q), an hypothesized side loop from G(1). All morphological stages, not only division, appear to be phased by the circadian clock.

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Cycloheximide, an inhibitor of protein synthesis on cytoplasmic ribosomes in eukaryotes, is shown to shift the phase of the circadian rhythm in stimulated bioluminescence in the marine dinoflagellate Gonyaulax polyedra. Kinetic analysis of the phase changes shows that this effect may be subdivided into two distinctly different and well-separated parts. The first (early) phase change occurs with 15-min exposure to cycloheximide and is saturated at low concentrations ( approximately 10 nM). The second (late) phase changes requires about 150 min of exposure to cycloheximide and is saturated at 0.36 muM cycloheximide. Twenty-times-higher concentrations cause no further phase changes. The magnitudes of both early and late phase changes depend on the time of day when the cells are exposed to cycloheximide. Early phase changes vary from 5 hr advance at circadian time (CT) 20 to 1 hr delay at CT 12; late phase changes are larger, the maximal advance being 12 hr at CT 16 and the greatest delay, 10 hr at CT 14. It is proposed that the early phase changes are caused by alterations in the ion distribution across membranes as a consequence of the permeation of cycloheximide. Late phase changes may be the result of inhibition of protein synthesis.The phase response curve for the late phase change is identical to that obtained with saturating light pulses in otherwise constant darkness in Gonyaulax. Maximal phase changes drive the clock into the part of the circadian cycle between CTs 4 and 9. Perturbations in this part of the circadian cycle are without effect on phase. Incubation of Gonyaulax with cycloheximide for a critical duration at a critical time induces arhythmicity, but longer exposures to the inhibitor at the same time do not. This observation suggests the existence of a singularity in the circadian clock of Gonyaulax.

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Circadian rhythms in acid-stimulated bioluminescence and cell division are observed for at least 16 days in bright continuous light (4.5 milliwatts per square centimeter or 20,000 lux). The photosynthesis rhythm also fails to stop immediately upon transfer of cell suspensions to bright light. After about 4 weeks under these conditions, all rhythms were observed to damp out. In cells transferred from bright light to continuous darkness, the rhythms were reset to about circadian hour 12 to 14, the phase of the beginning of a normal night.

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Exposure for 4 hours to vanillic acid (4-hydroxy 3-methoxy benzoic acid) caused large delay phase shifts (5 to 6 hours) in the circadian rhythm of bioluminescence in Gonyaulax polyedra, when assayed at either 10 to 14 circadian time or 22 to 02 circadian time in constant light and temperature, provided that the pH of the medium was 7.1 or lower. Corresponding changes in the pH with acetic acid did not shift phase. Vanillic acid caused detectable depolarization of the membranes of Gonyaulax, as demonstrated with the cyanine dye fluorescence technique.

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Circadian rhythms in photosynthesis were defined for the first time in the dinoflagellates Glenodinium sp. (M. Bernard strain) and Ceratium furca Ehrenberg (B. Meeson strain) and compared with that in Gonyaulax polyedra Stein. All three phytoplankton species had photosynthetic rhythms with daily amplitudes ranging from 3 to 5 and maxima displayed about midday. The photosynthetic pigment content and absorption properties of the cells were constant over the circadian cycle. Diurnal periodicities in respiration never accounted for more than 30% of the photosynthetic rhythm and did not persist under constant conditions. There was sufficient similarity between the circadian rhythms of these three dinoflagellates to suggest the mechanism of regulation may be the same for each of them.

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Using data from light-dark cultures of Gonyaulax polyedra entrained to a 24-hour cycle, whole cell absorption curves and photosynthesis-irradiance curves were constructed for various circadian times. While whole cell absorbance and half-saturation constants of photosynthesis showed no statistical difference that could be directly related to the photosynthetic rhythm, the initial slope of the photosynthesis-irradiance curve was a time-dependent parameter which altered in direct proportion to the change in photosynthetic capacity. The results indicated a temporal change in the relative quantum yield of photosynthesis, and the circadian rhythmicity of light-limited photosynthesis was established under constant conditions. Circadian rhythmicity was detected in room temperature chlorophyll fluorescence yield. Low temperature fluorescence kinetics also showed fluctuations. The results suggest that regulation of photosynthesis by the biological clock of Gonyaulax may be mediated through the membrane-bound light reactions and a partial explanation of the underlying mechanism is proposed.

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Intramembrane faces were visualized in the marine dinoflagellate Gonyaulax polyedra by the freeze-fracture technique, in order to test a prediction of a membrane model for circadian oscillations--i.e;, that membrane particle distribution and size change with time in the circadian cycle. Cells from each of four cell suspensions in continuous light (500 1x, 20-21 degrees C) were frozen, without fixation or cryoprotection, at four circadian times in a cycle. This paper reports findings concerning the membranes associated with the theca, particularly the cytoplasmic membrane and the membrane of the large peripheral vesicle. While the number and size distribution of the particles of the PF face of the cytoplasmic membrane were constant with time, those of the EF face of the peripheral vesicle doubled in number at 18 h circadian time as compared with 06 h. Particles of the 120-A size class, in particular, were more numerous at 12 and 18 h circadian time than at 00 and 06 h. While the finding does not provide definitive confirmation of the membrane hypothesis for circadian rhythms, it is consistent with this model. It is suggested that the peripheral vesicle may be the site of bioluminescence in Gonyaulax.

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Viable spheroplasts of a marine dinoflagellate, Gonyaulax polyedra, have been prepared for the first time. This simple and rapid procedure results in a yield of over 95% intact spheroplasts. Utilizing this technique, many studies of the cell-wall-free form of this dinoflagellate are now possible.