Recent progress in understanding the temporal behavior of unicellular organisms.

This survey summarizes the findings concerning endogenous oscillations of three unicellular organisms: the dinophyte Gonyaulax polyedra, the ciliate Tetrahymena thermophila and the euglenophyte Euglena gracilis. All of them behave rhythmically and show the common features of zeitgeber action, differential sensitivity and temperature compensation; however, they exhibit some species-specific peculiarities that make each of them suitable for addressing particular chronobiological questions. Although ultradian rhythms have been described for Tetrahymena thermophila and Euglena gracilis, they appear under different conditions: in the first case, a modulation of the period in relation to the concentration of nutrients is observed, whereas Euglena oscillates in an ultradian and circadian fashion simultaneously. Transitions between periodic and aperiodic states can be induced in Euglena gracilis and Gonyaulax polyedra: Euglena gracilis can enter an aperiodic state after repeated exposure to short light pulses (up to 10 sec) given at intervals of 40 min or less, whereas in Gonyaulax polyedra the circadian oscillator is arrested at temperatures below 12 degrees C. In the arrhythmic state, the oscillator might be driven into singularity within the phase space of a limit cycle attractor; re-initiation from the holding point occurs by transition to a relatively precisely defined new phase. Photoperiodism as another important chronobiological phenomenon can be studied in Gonyaulax polyedra: cells enter the dormant stage of an asexual cyst under short days and a temperature below 16 degrees C. This response can be mimicked by 5-methoxylated indoleamines such as melatonin and 5-methoxytryptamine, which are synthesized by this organism. Melatonin concentration exhibit an endogenous circadian rhythm characterized by a rapid increase shortly after the onset of darkness. Encystment, as induced by indoleamines, is associated with stimulations of bioluminescence. The coupling of the two processes involves, as a common element, the release of protons from an acidic vacuole.

Recent progress in understanding the temporal behavior of unicellular organisms

This survey summarizes the findings concerning endogenous oscillations of three unicellular organisms: the dinophyte Gonyaulax polyedra, the ciliate Tetrahymena thermophila and the euglenophyte Euglena gracilis. All of them behave rhythmically and show the common features of zeitgeber action, differential sensitivity and temperature compensation; however, they exhibit some species-specific peculiarities that make each of them suitable for addressing particular chronobiological questions. Although ultradian rhythms have been described for Tetrahymena thermophila and Euglena gracilis, they appear under different conditions: in the first case, a modulation of the period in relation to the concentration of nutrients is observed, whereas Euglena oscillates in an ultradian and circadian fashion simultaneously. Transitions between periodic and aperiodic states can be induced in Euglena gracilis and Gonyaulax polyedra: Euglena gracilis can enter an aperiodic state after repeated exposure to short light pulses (up to I 0 sec) given at intervals of 40 min or less, whereas in Gonyaulax polyedra the circadian oscillator is arrested at temperatures below 12 degrees Celsius. In the arrhythmic state, the oscillator might be driven into singularity within the phase space of a limit cycle attractor; re-initiation from the holding point occurs by transition to a relatively precisely defined new phase. Photoperiodism as another important chronobiological phenomenon can be studied in Gonyaulax polyedra: cells enter the dormant stage of an asexual cyst under short days and a temperature below 16 degrees Celsius. This response can be mimicked by 5-methoxylated indoleamines such as melatonin and 5-methoxytryptamine, which are synthesized by this organism. Melatonin concentration exhibits an endogenous circadian rhythm characterized by a rapid increase shortly after the onset of darkness. Encystment, as induced by indoleamines, is associated with stimulations of bioluminescence. The coupling of the two processes involves, as a common element, the release of protons from an acidic vacuole.

On the primary functions of melatonin in evolution: mediation of photoperiodic signals in a unicell, photooxidation, and scavenging of free radicals.

Melatonin is widely abundant in many eukaryotic taxa, including various animal phyla, angiosperms, and unicells. In the bioluminescent dinoflagellate Gonyaulax polyedra, melatonin is produced in concentrations sometimes exceeding those found in the pineal gland, exhibits a circadian rhythm with a pronounced nocturnal maximum, and mimics the short-day response of asexual encystment. Even more efficient as a cyst inducer is 5-methoxyptryptamine (5MT), which is also periodically formed in Gonyaulax. In this unicell, the photoperiodic signal-transduction pathway presumably involves melatonin formation, its deacetylation to 5MT, 5MT-dependent transfer of protons from an acidic vacuole, and cytoplasmic acidification. According to this concept, we observe that cyst formation can be induced by various monoamine oxidase inhibitors and protonophores, that 5MT dramatically stimulates H(+)-dependent bioluminescence and leads to a decrease of cytoplasmic pH, as shown by measurements of dicyanohydroquinone fluorescence. Cellular components from Gonyaulax catalyze the photooxidation of melatonin. Its property of being easily destroyed by light in the presence of cellular catalysts may have been the reason that many organisms have developed mechanisms utilizing this indoleamine as a mediator of darkness. Photooxidative reactions of melatonin, as studied with crude Gonyaulax extracts and, more in detail, with protoporphyrin IX as a catalyst, lead to the formation of N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) as one of the main products. Photochemical mechanisms involve interactions with a photooxidant cation radical leading to the formation of a melatonyl cation radical, which subsequently combines with a superoxide anion.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of indoleamines and short photoperiods on the encystment of Gonyaulax polyedra.

At a temperature of 15 degrees C, Gonyaulax polyedra responds to short days (light less than or equal to 10 h) by transition to the stage of a resting cyst. At 20 degrees C, even an light:dark (LD) cycle of 6:18 is incapable of inducing this process. In otherwise cyst-inducing conditions (15 degrees C; 10 h of light per day), an interruption of the scotophase by 2 h of light (LDLD 8:2:2:12 or 2:2:8:12) prevented encystment. Cyst induction is, therefore, initiated by a photoperiodic mechanism rather than by light deficiency. In Gonyaulax, photoperiodism may be mediated by the action of indoleamines. Melatonin, which exhibits a circadian rhythmicity in this organism, leads to encystment when given 1 h before lights-off in LD 11:13 at 15 degrees C, i.e., under otherwise noninducing conditions. Again, at 20 degrees C, melatonin is inefficient. Some analogues of melatonin, in particular, 5-methoxytryptamine and N,N-dimethyl-5-methoxytryptamine, and, at high concentrations, their respective precursors, serotonin and bufotenin, are capable of inducing cyst formation at 20 degrees C and in LD 12:12, whereas N-acetyl-serotonin does not show this effect.

Photoperiodism and effects of indoleamines in a unicellular alga, Gonyaulax polyedra.

Mediation of photoperiodic effects by indoleamines, especially melatonin, is known in higher vertebrates. A similar mechanism may occur in a unicellular alga, the dinoflagellate Gonyaulax polyedra. This organism entered the dormant stage of a cyst upon short-day treatment at lowered temperatures. Interruption of darkness by 2 hours of light prevented cyst formation, even when the overall duration of light was the same as in cyst-inducing short days. When given in a noninducing photoperiod, melatonin and an analog, 5-methoxytryptamine, substances that had previously been shown to occur in Gonyaulax, provoked cyst formation. Methoxylated indoleamines may play a role as mediators of darkness in this unicellular, in a similar way as in vertebrates, suggesting a common biochemical basis of photoperiodism.

Circadian rhythmicity in the stimulation of bioluminescence by biogenic amines and MAO inhibitors in Gonyaulax polyedra.

In the dinoflagellate Gonyaulax polyedra, bioluminescence was investigated in constant darkness. Light emission was stimulated considerably and specifically by the biogenic amines epinephrine, 5-methoxytryptamine, and kynuramine. Various analogues and metabolites of these substances, such as norepinephrine, isoproterenol, phenylephrine, synephrine, metanephrine, dopamine, 3,4-dihydroxymandelic and 3-methoxy-4-hydroxymandelic acids, serotonin, N-acetylserotonin, melatonin, 5-hydroxytryptophol, 5-methoxytryptophol, kynurenine, 4-hydroxyquinoline, 3-hydroxyanthranilic, and quinolinic acids were much less effective. Strong enhancement of bioluminescence, in the range of those obtained with the three stimulatory biogenic amines, was also observed after administration of several compounds acting as MAO inhibitors in mammalian systems, in particular, pargyline, amitriptyline, p-benzoquinone, tranylcypromine, harmaline, and noreleagnine. The responsiveness of cells towards epinephrine, 5-methoxytryptamine, kynuramine, amitriptyline, p-benzoquinone, and noreleagnine varied considerably within the circadian cycle, with the highest stimulations obtained during subjective night. These rhythms can be only partially explained by periodic bioluminescence capacity, and seem to comprise a cyclicity in the sensitivity of cells to the compounds mentioned.

Temperature compensation in an ultradian rhythm of tyrosine aminotransferase activity in Euglena gracilis Klebs.

Tyrosine aminotransferase activity of Euglena oscillates with an ultradian period of approximately 4-5 h. The oscillation frequency in the time series was determined by cosine fitting. Experiments which were performed between 16 and 31.5 degrees C revealed temperature compensation.

[Immunohistochemical tumor diagnosis in breast cancer--use for assessing the stage and biology of so-called "small breast cancer"]. / Immunhistochemische Tumordiagnostik beim Mammakarzinom--Einsatz zur Evaluierung von Stadium und Biologie des sogenannten "kleinen Mammakarzinoms".

Immunohistochemical methods assure the discrimination between neoplastic and reactive Lesions. Epithelial and mesenchymal neoplastic processes can absolutely be distinguished by antibodies directed to intermediate filaments. Growth activity of breast cancer can be evaluated using antibodies against epidermal growth factor receptor and Ki-67 proliferative protein. With antibodies directed to nuclear steroid receptor proteins semiquantitative determinations (immunoreactive score = IRS) and evaluation of genotypic heterogeneity are possible and may be helpful for therapeutic decisions.

Mitochondrial acetyl-CoA acetyltransferase in liver and extrahepatic tissues: role of modification by coenzyme A.

The influence of clofibrate and di(2-ethylhexyl)phthalate on mitochondrial acetyl-CoA acetyltransferase (acetyl-CoA: acetyl-CoA C-acetyltransferase, EC 2.3.1.9), the rate-limiting ketogenic enzyme, which can be modified and inactivated by CoA, was investigated. In fed rats, both compounds induced a doubling of ketone bodies in the blood and, moreover, an increase by about 13% in the hepatic relative amount of the unmodified, i.e., the most active form of the enzyme (immunoreactive protein). This shift would account for an elevation of overall enzyme activity by about 5% only. Thus, the CoA modification of mitochondrial acetyl-CoA acetyltransferase did not explain the entire augmentation of ketone bodies. However, clofibrate and di(2-ethylhexyl)phthalate also increased the immunospecific protein and enzyme activity by approx. 2- and 3-fold, respectively. These effects were observed in liver, but not in several extrahepatic tissues.

Rapid thin layer chromatographic method for determining aflatoxin B1, ochratoxin A, and zearalenone in corn.

A multimycotoxin thin layer chromatographic method is described for the analysis of corn. Aflatoxins are extracted from the samples with acetonitrile-water, and sodium bicarbonate is added to separate the acidic ochratoxin from zearalenone and aflatoxin B1. After chloroform extraction, 1N NaOH is added to separate zearalenone and aflatoxin B1. The separated mycotoxins are spotted on TLC plates, which are then examined under ultraviolet light. The following recoveries (%) were obtained for corn samples: aflatoxin B1 71, ochratoxin A 87, and zearalenone 85. The limits of detection for the respective mycotoxins were 2, 40, and 200 ppb.

Natural contamination of corn (Zea maïs) with mycotoxins in Yugoslavia.

The 191 corn samples were collected in autumm and spring following the crop year 1975 from terminal elevators and from silos during the storage, at feed processing establishment. Some samples were taken on the farms at the region "Posavina" where the "Balkan kidney disease"--endemic nephrytis--is wide spread. The samples were analysed for : zearalenone, aflatoxins, skin irritating factors and ochratoxin A, by a modified multimycotoxins method. Zearalenone was found in 2.6 p. 100 samples (0.043-10 ppm), average 5.1 ppm. Aflatoxins were found in no samples. A skin irritating factor was found in 8.3 p. 100 samples (0.5-5.0 ppm), average 2.2 ppm, and ochratoxin A was found in 26 p. 100 samples (0.045-5.1 ppm), average 0.49 ppm.