Recording the oxygen production of a single Acetabularia cell for a prolonged period

A method for recording the O₂ evolution of an individual Acetabularia cell or cell fragment over a period of weeks is described. The method is based on the polarographic O₂ determination by means of a platinum electrode in a flow-through system. The mean O₂ evolution of a full-grown cell under constant conditions (2 5001m/m², 20 °C) was 3–6 μ1 O₂ per cell perh. Under these conditions the O₂ evolution exhibited a pronounced circadian rhythm with an average period of about 23 h and an amplitude of about 2.3 μ1 O₂ per cell per h. No significant differences were found between nucleate and anucleate cells.     

Studies on Independent Synthesis of Cytoplasmic Ribonucleic Acids in Acetabularia mediterranea

1. The RNA content of anucleate and nucleate fragments of Acetabularia has been measured. It was found that there is a net synthesis of RNA in nucleate fragments. On the other hand, the RNA content of anucleate fragments did not change significantly after enucleation. 2. Anucleate fragments, however, can readily incorporate ¹⁴C-labeled adenine, orotic acid, and carbon dioxide into their cytoplasmic RNA. 3. The results of experiments on ¹⁴CO₂ incorporation into the RNA of anucleate and nucleate fragments suggest that there is a mechanism for de novo synthesis of RNA in anucleate cytoplasm. 4. In Acetabularia, 81 per cent of the cytoplasmic RNA is bound to a large granule fraction, consisting mainly of chloroplasts. Even after removal of the nucleus, RNA is synthesized in this "chloroplast" fraction. The chloroplasts are thus a major site of RNA synthesis in the cytoplasm of these algae. Synthesis of "chloroplastic" RNA, in anucleate fragments, possibly occurs at the expense of the RNA present in other fractions (microsomes and supernatant). 5. 8-Azaguanine stimulates regeneration and cap formation in anucleate fragments and does not inhibit RNA synthesis in these fragments.     

Circadian properties of the rhythmic system in individual nucleated and enucleated cells of Acetabularia mediterranea.

The method for the continuous polarographic monitoring of O₂ has been used for systematic studies of the phase and period of the circadian rhythm of photosynthetic O₂ production in individual Acetabularia cells with and without nuclei. The period of the rhythm is highly variable and is modulated by an effective, but imperfect temperature-compensating system which makes the period longer at higher temperatures. Changes in the phase of the free-running rhythm can be effected by single brief dark pulses administered during characteristic portions of the cycle. The equivalence of the dark pulse response curves and temperature coefficients in nucleate and enucleate cells reaffirms the cytoplasm's pronounced capacity for autonomous circadian regulation.     

Stability of poly(A)+RNA in nucleate and anucleate cells of Acetabularia

The stability of poly(A)⁺RNA was compared in nucleate and anucleate cells of Acetabularia. While in the absence of the nucleus poly(A)⁺RNA exhibits a pronounced stability, it is significantly less stable in the presence of the nucleus.     

Kernkontrollierte Lactatdehydrogenase inAcetabularia

Zusammenfassung In kernhaltigen, aber auch in kernlosen Zellen vonAcetabularia wird wÄhrend des Wachstums die LDH-AktivitÄt bis zum Beginn der Hutbildung vermehrt. Verschiedene Arten vonAcetabularia weisen bezüglich der LDH entweder durch Zahl der Banden oder Position der Banden Unterschiede voneinander auf. Heterologe Transplantate bzw. Implantate lassen einige Wochen nach der Operation erkennen, da\ die artspezifischen Isozymbanden des Cytoplasmas verschwinden und die vom Kern determinierten erscheinen. Zumindest ein Teil der LDH-AktivitÄt ist an die Chloroplasten gebunden. Dieses wurde mit Hilfe der Saccharose-Dichtegradienten-Zentrifugation und auf histochemischem Wege nachgewiesen.

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Nucleus dependent lactic dehydrogenase inAcetabularia

Summary In nucleate as well as in anucleate cells ofAcetabularia LDH activity is increased until the cap is formed. Different species ofAcetabularia can be distinguished by the electrophoretic behavior of the LDH. Heterologous grafts of nucleus containing rhizoids or of isolated nuclei to anucleate cytoplasm result in the disappearance of the LDH pattern of the cytoplasm and in the appearance of a new one which is specific for the species of the nucleus. At least part of the enzyme activity is linked to the chloroplasts as was shown by sucrose density gradient centrifugation and by histochemical techniques.

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Wir danken Herrn Dr. G.Werz für Anregungen beim histochemischen Nachweis und Herren H.Kretschmer für seine Hilfe bei der PrÄparation der Transplantate und Implantate.     

Differenzierung und Zellwandbildung in isoliertem Cytoplasma ausAcetabularia

Zusammenfassung Die vorliegende Arbeit beschreibt die Isolierung kernhaltigen und kernfreien Cytoplasmas aus den Hutkammern vonAcetabularia und dessen morphogenetisches Verhalten unterin vitro-Bedingungen.Kernhaltiges Cytoplasma (Protoplasten) bildet stets Cysten und cystenspezifische Zellwand.Die Fähigkeit kernlosen Cytoplasmas (Cytoplasten) zur Differenzierung und zur Synthese von Cystenwand hängt von der Einwirkungszeit des Kernes vor der Isolierung ab. Daher zeigen Cytoplasten aus Hutkammern, die keine Kerne enthielten, weder Differenzierung noch Bildung von Cystenwand. Differenzierung und Bildung von Cystenwand findet in gewissem Ausmaß dann statt, wenn Cytoplasten aus Hutkammern isoliert werden, in denen das Cytoplasma unter Kerneinfluß einen höheren Grad der Differenzierung erreicht hatte.

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Differentiation and cell wall formation in isolated cytoplasm ofAcetabularia

Summary The present paper reports on the isolation of nucleate and anucleate cytoplasm of cap rays ofAcetabularia and with its morphogenetic behaviour underin vitro conditions.The nucleate cytoplasm (protoplasts) always differentiates cysts and cyst specific cell wall.The ability of anucleate cytoplasm (cytoplasts) to differentiate and to synthesize cyst wall depends on the time of nuclear actions prior to its isolation. Therefore, cytoplasts of cap rays which did not contain nuclei, show neither differentiation nor cyst wall formation. A certain degree of differentiation and of cyst wall formation takes place if cytoplasts are isolated from cap rays in which the cytoplasm had obtained a higher degree of differentiation eunder nuclear actions.

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Fräulein R.Sandfuchs danke ich für die Aufzucht der Versuchspflanzen, Herrnstud. rer. nat. K.Ertl für seine Hilfe bei der Durchführung der Experimente.     

Ribonuclease-Aktivität in Acetabularia

Zusammenfassung In Acetabularia mediterranea, Acetabularia crenulata und Polyphysa cliftonii wurde das Vorhandensein von Ribonuclease-Aktivität nachgewiesen. Es wurde gezeigt, daß das Enzym hitzestabil ist und bei pH 7,2 und im Bereich von pH 5 Maxima aufweist. Selbst durch eine Zentrifugation nit 105000 x g über 120 min wurde die Enzym-Aktivität nicht sedimentiert.In kernlosen Zellen von Acetabularia nimmt die Ribonuclease-Aktivität zu, allerdings in geringerem Maße als in kernhaltigen Zellen.Die Stabilität der genetischen Information im Cytoplasma von kernloser Acetabularia kann nicht durch das Fehlen von Ribonuclease-Aktivität erklärt werden.Fräulein A. Grohs sei für die geschickte und sorgfältige Hilfe bei der Durchführung der Experimente gedankt.

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Activity of ribonuclease in Acetabularia

Summary In Acetabularia mediterranea, Acetabularia crenulata and Polyphysa cliftonii ribonuclease activity was demonstrated. The enzyme is heat stabile and exhibits maxima at pH 7.2 and at the region of pH 5. Even centrifugation with 100000 x g for 120 minutes does not result in sedimentation of enzyme activity.Anucleate cells of Acetabularia are capable of increasing their ribonuclease activity, but to a smaller extent than nucleate cells do.The stability of the genetic information in the cytoplasm of anucleate Acetabularia cannot be explained by a lack in ribonuclease activity.

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Herrn Prof. J. Hämmerling zum 65. Geburtstag gewidmet.     

Circadian photosynthetic reductant flow in the dinoflagellate Lingulodinium is limited by carbon availability

Circadian rhythms are the observed outputs of endogenous daily clocks and are thought to provide a selective advantage to cells adapted to daily light/dark cycles. However, the biochemical links between the clock and the overt rhythms in cell physiology are generally not known. Here, we examine the circadian rhythm in O₂ evolution by cultures of the dinoflagellate Lingulodinium, a rhythm previously ascribed to rhythmic electron flow through photosystem II. We find that O₂ evolution rates increase when CO₂ concentrations are increased, either following addition of DIC or a rapid decrease in culture pH. In medium containing only nitrate as an electron acceptor, O₂ evolution rates mirror the circadian rhythm of nitrate reductase activity in the cells. Furthermore, competition between photosynthetic electron flow to carbon and to nitrate varies in its relative efficiency through the day–night cycle. We also find, using simultaneous and continuous monitoring of pH and O₂ evolution rates over several days, that while culture pH is normally rhythmic, circadian changes in rates of O₂ evolution depend not on the external pH but on levels of internal electron acceptors. We propose that the photosynthetic electron transport rhythm in Lingulodinium is driven by the availability of a reductant sink.     

A method for recording the circadian rhythm of the oxygen balance in a single cell ofAcetabularia mediterranea

Summary A polarographic method using a platinum electrode permits continuous recording of the oxygen balance of a singleAcetabularia cell during several days. The amount of oxygen which is consumed by the electrode is immeasurably small. The accuracy of the method is definitely better than 0.05l O₂/ml medium. The resolution in time is less than 5 min. The length of one period in the circadian rhythm of photosynthesis inAcetabularia has been found to be about 27 hours.     

Temperature Dependency of Circadian Period in a Single Cell (Acetabularia)

The circadian rhythm in the oxygen production of 30 individual Acetabularia cells has been studied at different temperatures. The temperature induced period variation was continuously evaluated over the whole data record of each individual cell with an advanced spectral analysis technique. The observed circadian periods of O₂ production displayed a well established region of temperature compensation between 25 °C and 30 °C with a Q₁₀, value of 0.9, whereas between 15°C and 22°C a positive temperature coefficient was measured (Q₁₀ at 22 °C 0.9, Q₁₀ at 20°C 0.8, Q₁₀at 17°C 0.7).     

Limitations in chloroplast multiplication inAcetabularia mediterranea

Summary The incorporation of thymidine into chloroplastic DNA inAcetabularia, examined two times after enucleation, has been compared with known data on the evolution of the DNA content of these organelles after enucleation.Division of the chloroplasts has been examined in cytoplasts obtained from intact and anucleate algae and compared with known data on chloroplast division in algae enucleated since various periods of time (up to three weeks) and with corresponding electron micrographs.The distribution curves of the chloroplast size in intact algae, anucleate fragments, and cytoplasts were established.On the basis of the experimental evidence, it is proposed that both replication of chloroplastic DNA and division of the chloroplasts are under nuclear control but that information or factors involved in the former have a longer life time than those involved in the latter.In addition, some factors involved in division seem to be located in the peripheral part of the cytoplasm ofAcetabularia. Alternatively, they could be lost by the cytoplasts.     

Auslösung der circadianen Photosynthese-Rhythmik beiAcetabularia durch Blaulicht

Summary Additional irradiation with blue light of low intensity ofAcetabularia mediterranea cells, pretreated by prolonged irradiation with red, induces an increase of photosynthetic activity. This induction is accompanied by the appearance of rhythmic changes of the O₂ production. Maxima are found about 6, 30, 54, 78, ... hours after the onset of blue light irradiation. Thus blue light not only induces an increase of the rate of photosynthesis but also acts as a Zeitgeber for the circadian rhythm of photosynthesis inAcetabularia.

     

Regulation of the Photosynthesis Rhythm in Euglena gracilis: I. Carbonic Anhydrase and Glyceraldehyde-3-Phosphate Dehydrogenase Do Not Regulate the Photosynthesis Rhythm

A circadian rhythm of O₂ evolution has been found in Euglena gracilis, Klebs strain Z. The rhythm persists for at least 5 days in constant dim light and temperature, but damps out in constant bright light. The phase of this rhythm can be shifted by a pulse of bright light and the period length is not changed over a 10 C span of growth temperature.

The O₂ evolution rhythm is found in both logarithmic and stationary phase cultures, but CO₂ uptake is clearly rhythmic only in stationary phase cultures.

The activity of glyceraldehyde-3-phosphate dehydrogenase was not rhythmic as previously reported (Walther and Edmunds [1973] Plant Physiol. 51: 250-258). Carbonic anhydrase activity was rhythmic when the cultures were maintained under a light-dark cycle with the highest enzyme activity coinciding with the fastest rate of O₂ evolution. However, the rhythm in carbonic anhydrase activity disappeared under constant conditions. Changes in the activities of these two enzymes are therefore not responsible for the rhythmic changes in photosynthetic capacity.

     

Twofold effect of blue light on a circadian rhythm in Acetabularia

Abstract

The circadian chloroplast migration in Acetabularia mediterranea was monitored by continuously measuring the transmission of the cells near the apex. Under continuous red light the amplitude of the rhythm decreased rapidly within a few days. However, circadian changes of chloroplast density were still detectable even after 28 days of red light, indicating the persistence of the rhythm. When blue light was added after red light preirradiation of several days phase shifts were observed which were expressed as advances as well as delays. The period of the rhythm proved to be strongly dependent on the intensity of the continuous blue light which was given in addition to red light. Different red light intensities did not change the period. The occurrence of both effects indicates that the sensory transduction of blue light photoreception in Acetabularia works in two different ways: quanta counting processes and processes of light intensity measurement.     

Die Wirkung von Rifampicin auf die RNA- und Proteinsynthese sowie die Morphogenese und den Chlorophyllgehalt kernhaltiger und kernloser Acetabularia-Zellen

Summary Rifampicin (10g/ml) strongly inhibits the incorporation of [5-³H]-uridine into the chloroplast RNA of anucleate cells of the green alga Acetabularia mediterranea, whereas incorporation into nuclear RNA is hardly affected.Furthermore, at a concentration range of 1–10g/ml rifampicin has only a small effect on stalk- and cap formation in nucleate posterior parts of the stalk. As has already been shown, the morphogenesis of such cell segments depends on the synthesis of new RNA in the nucleus. Similarly rifampicin only slightly inhibits the synthesis of the enzyme UDPG-pyrophosphorylase, which is coded by nuclear DNA.These slight inhibitions are interpreted as secondary effects arising from a blockage of plastid RNA synthesis, since both nucleate and anucleate cells respond in a similar manner and to the same degree.In contrast the increase in the chlorophyll content in nucleate and anucleate cells is severely impaired by the antibiotic. These findings indicate that the nucleus and the plastids contain different DNA-dependent RNA-polymerases.     

Elektronenmikroskopische Untersuchungen zur Genese des Golgi-Apparates (Dictyosomen) und ihrer Kernabhängigkeit beiAcetabularia

Summary The genesis of dictyosomes has been studied on nucleate and enucleateAcetabularia cells.Dictyosomes differentiatede novo from prestages, the perinuclear bodies, which are synthesized under the actions of nuclear ribonucleoprotein. Consequently the formation of dictyosomes is nucleus (DNA) dependent.This was shown by experiments using actinomycin and puromycin and by experiments on darkened and enucleate cells.There is a relationship between dictyosome structure and function and the morphogenetic activities of a cell.

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Mit 10 Textabbildungen     

Polarity of Acetabularia mediterranea: Stability in the Anucleate State

Concomitant with the outgrowth of the cap in cells of Acetabulariamediterranea Lamouroux, the stalk acquires a pale green appearance(‘Aufhellung’) and the chlorophyll and protein contentdecreases due to the translocation of part of the cytoplasmfrom the stalk into the cap. This process also occurs in anucleatecells or apical fragments during cap formation and in inversegrafts constructed either from two anucleate cells or apicalfragments or from two basal fragments containing the rhizoids.The cytoplasm's translocation does not depend on the presenceof the nucleus. It is suggested that the unidirectional flowof the cytoplasm during cap formation is generated by the electricalcharge across the cell and that this electrical charge is maintainedafter removal of the nucleus. Acetabularia, polarity, anucleate cells, grafts     

Novel S-Benzylisothiourea Compound That Induces Spherical Cells in Escherichia coli Probably by Acting on a Rod-shape-determining Protein(s) Other Than Penicillin-binding Protein 2

Random screening for inhibitors of chromosome partitioning in Escherichia coli was done by the anucleate cell blue assay. A novel S-benzylisothiourea derivative, S-(3,4-dichlorobenzyl)isothiourea, tentatively named A22, was found to induce spherical cells and spherical anucleate cells in E. coli. Mecillinam, a specific inhibitor of penicillin-binding protein 2, which induces spherical cells in E. coli, also caused anucleate cell production. Spherical cells induced by treatment with either A22 or mecillinam varied in size, and anucleate cells seemed to be more frequent among the smaller cells. These results suggest that loss of the rod shape in E. coli leads to asymmetric cell division that results in production of anucleate cells. No competition was observed even in the presence of a 10-fold excess A22 in an in vitro assay of ¹⁴C-penicillin G binding, but mecillinam specifically inhibited binding of ¹⁴C-penicillin G to penicillin-binding protein 2. Simultaneous treatment with mecillinam and cephalexin, a specific inhibitor of penicillin-binding protein 3, induced lysis of E. coli cells, but a combination of A22 and cephalexin did not. These results suggest that the target molecule(s) of A22 was not penicillin-binding protein 2. A22 may act on a rod-shape-determining protein(s) other than penicillin-binding protein 2, such as RodA or MreB.     

Metabolic stability and inhibitory effect of O-methylated theaflavins on H2O2-induced oxidative damage in human HepG2 cells

Seven new O-methylated theaflavins (TFs) were synthesized by using O-methyltransferase from an edible mushroom. Using TFs and O-methylated TFs, metabolic stability in pooled human liver S9 fractions and inhibitory effect on H₂O₂-induced oxidative damage in human HepG2 cells were investigated. In O-methylation of theaflavin 3′-O-gallate (TF3′G), metabolic stability was potentiated by an increase in the number of introduced methyl groups. O-methylation of TF3,3′G did not affect metabolic stability, which was likely because of a remaining 3-O-galloyl group. The inhibitory effect on oxidative damage was assessed by measuring the viability of H₂O₂-damaged HepG2 cells treated with TFs and O-methylated TFs. TF3,3′G and O-methylated TFs increased cell viabilities significantly compared with DMSO, which was the compound vehicle (p?<?0.05), and improved to approximately 100%. Only TF3′G did not significantly increase cell viability. It was suggested that the inhibitory effect on H₂O₂-induced oxidative damage was potentiated by O-methylation or O-galloylation of TFs.     

Photosynthetic characteristics of coccoid marine cyanobacteria

Two strains of marine Synechococcus possessed a much greater potential for photorespiration than other marine algae we have studied. This conclusion was based on the following physiological and biochemical characteristics: a) a light-dependent O₂ inhibition of photosynthetic CO₂ assimilation at atmospheric O₂ concentrations. The degree of inhibition was dependent on the relative concentrations of dissolved O₂ and CO₂, being greatest at 100% O₂ with no extra bicarbonate added to the medium; b) actively photosynthesizing cells had high levels of ribulose-1,5-bisphosphate carboxylase compared with phosphoenolpyruvate carboxylase; ribulose-1,5-bisphosphate oxygenase activities were three times greater than ribulose-1,5-bisphosphate carboxylase activities; c) cells photosynthesizing in 21% O₂, showed significant ¹⁴C-labelling of phosphoglycolate and glycolate and the percentage of total carbon-14 incorporated into these two compounds increased when the O₂ concentration was 100%; d) at 100% O₂, there was a post-illumination enhanced rate of O₂ consumption, which was three times greater than dark respiration, and the rate declined with increasing bicarbonate concentrations. The inhibitory effect of O₂ on photosynthesis did not appear to be solely due to photorespiration, since O₂ inhibition of photosynthetic O₂ evolution was much greater than that of photosynthetic CO₂ assimilation. Also, O₂ inhibition of photosynthetic O₂ evolution declined only slightly with decreasing light intensities, while the inhibition of CO₂ assimilation declined rapidly with decreasing light intensity.