Studies on the vegetative life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture I. Some characteristics of the cell cycle

Cells of Chlamydomonas reinhardi Dangeard were grown synchronouslyunder a 12 hr light-12 hr dark regime. Time courses of nucleardivision, chloroplast division, "apparent cytokinesis" and zoosporeliberation were followed during the vegetative cell cycle inthe synchronous culture. Liberation of zoospores occurred atabout 23–24 hr after the beginning of the light periodat 25°C. Four zoospores were produced per mother cell underthe conditions used. At lower temperatures, the process of zoosporeliberation as well as length of the cell cycle was markedlyprolonged, but the number of zoospores produced per mother cellwas approximately the same. At different light intensities,lengths of the cell cycle were virtually the same, while thenumber of zoospores liberated was larger at higher rather thanat lower light intensities. During the dark period, nuclear division, chloroplast divisionand apparent cytokinesis took place, in diis order, and proceededless synchronously than did the process of zoospore liberation.When the 12 hr dark period was replaced with a 12 hr light periodduring one cycle, the time of initiation as well as the durationof zoospore liberation was litde affected in most cases, whereasnuclear division, chloroplast division and apparent cytokinesiswere considerably accelerated by extended illumination. Whenalgal cells which had been exposed to light for 24 hr were furtherincubated in the light, zoospore liberation started much earlierand proceeded far less synchronously, compared with that under12 hr light-12 hr dark alternation. (Received October 12, 1970; )     

Studies on the vegetative life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture IV. Effects of 6-methyl purine on the revolution of the cell cycle

Cells of Chlamydomonas reinhardi Dangeard were synchronouslygrown under a 12 hr light-12 hr dark regime. The algal cellcycle under these conditions starts with a light-induced reaction(s)at the beginning of the light period and ends, after a definiteperiod of time (23–24 hr at 25°C), in zoospore liberation.When cells were exposed to 6-methyl purine for short periods(0.5–2.5 hr) at different times during the early and intermediatephases of the cell cycle, it exerted, as an analogue of adenine,two different effects on the revolution of the cell cycle: onea "lengthening" effect seen at its low concentrations in whichthe length of the cell cycle was somewhat prolonged, the othera "return to start" effect at higher concentrations. In thelatter a short exposure of cells to 6-methyl purine broughtthem to the starting point of the cell cycle concurrent withthe abortion of the cycle in process. When 6-methyl purine wasapplied during the later phase of about 1/4 the length of thecell cycle, it casued no effect. Control of the revolution ofthe algal cell cycle by an "adenine-involving reaction(s)" disturbedby this adenine analogue is discussed. (Received September 1, 1975; )     

Studies on the vegetative life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture V. The transition point for the effect of 6-methyl purine in the cell cycle

By following the nuclear division, chloroplast division, cytokinesisand zoospore liberation of Chlamydomonas reinhardi cells exposedto a high concentration of 6-methyl purine (6-MP), corroborativeevidence was obtained for our previous conclusion that 6-MP-exposedcells are brought to the starting point of a new round of thecell cycle with abortion of the cycle in process ("return-to-start"effect). This effect did not occur after cells had passed acritical stage (transition point) which seemed to be situatedshortly prior to the onset of nuclear division under the conditionsused. When 6-MP was applied to cells after the transition point,it caused an advancing effect on their zoospore liberation.A cycloheximide (CHI)-inhibition step existed shortly alterthe transition point for 6-MP. A model was proposed for theeffects of 6-MP and CHI. (Received August 8, 1977; )     

Studies on the vegetative life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture II. Effects of chloramphenicol and cycloheximide on the length of cell cycle

Cells of Chlamydomonas reinhardi Dangeard were synchronouslygrown under a 12 hr light— 12 hr dark regime. When thesecells were brought into contact with chloramphenicol for a shortperiod at early stages in the cell cycle, zoospore liberationwas delayed for a period which was nearly the same as that ofthe duration of contact with the antibiotic. When given at laterstages, the antibiotic caused no such effect. Cycloheximide,on the other hand, caused—when provided at some intermediatestage of the cell cycle— two different prolonging effectson the length of the cell cycle: one doubled the normal length(observed when the drug was administered at certain stages)and the other caused a delay similar to that caused by chloramphenicol.Interestingly, no prolonging effect was observed when cycloheximidewas given either at early stages or at later stages, such asduring the last 1/4 period of the cell cycle preceding zoosporeliberation. Based on these results, three phases were distinguishedin the algal cell cycle: "chloramphenicolsensitive", "cycloheximide-sensitive"and "insensitive" phases. Considering the known facts aboutthe modes of action of the two antibiotics inhibiting proteinsynthesis, discussions were made on the significance of proteinsynthesis in chloroplasts and in cytoplasm in determining thelength of the cell cycle. (Received October 12, 1970; )     

A short note on the effects of 6-methyl purine on the revolution of the cell cycle of Chlorella pyrenoidosa in synchronous culture

Short exposure of Chlorella pyrenoidosa cells to a high concentrationof 6-methyl purine atan early stage of the cell cycle causedan apparent "return-to-start" effectin which the cellsseemedto return to the starting point of a new cell cycle, with concurrentabortion of the cell cycle in process. (Received November 25, 1975; )     

Infection of the diatom Asterionella by a chytrid. I. Effects of light on reproduction and infectivity of the parasite

The influence of hight limitation of the diatom Asterionellaformosa Hass, on the growth-determining parameters of its fungalparasiteRhizophydium planktonicum Canter emend, was measured,using laboratory cultures of both organisms. The experimentswere earned out at 6°C under a 15:9 h light-dark cycle.At saturating light conditions, the mean zoospore productionof the parasite was 23.4 zoospores sporangium^–1, and themean development time of the sporangia was 7 9 days. Light limitationof the host caused a substantial decrease of the zoospore production,while the development time was only slightly reduced. The improvedzoospore production at high light intensities was mainly theresult of incorporation of photosynthetic products generatedby the host after infection. Under limiting light conditions,Asterionella cells were less susceptible to infection withfungal zoospores. No infection at all occurred below 2 µEm^–2 s^–1, a light intensity that still supportedsome algal growth The maximum infection rate indicated thatchemotactic attraction of the parasite's zoospores by extracellularproducts of the host is involved. The infective lifetime ofthe zoospores of the parasite did not depend on light conditions,and was estimated at 8 days. The measured zoospore productionrates, both under limiting and saturating light conditions,enable the parasite to exceed the specific growth rate of thehost, and thus become epidemic, at sufficiently high host densities.     

Oligosaccharide side chains of wall molecules are essential for cell-wall lysis in Chlamydomonas reinhardtii

The glycoproteins of the cell walls of Chlamydomonas are lysed during the reproductive cycle by proteases (autolysins) which are specific for their substrates. The autolysin which digests the wall of sporangia to liberate the zoospore daughter cells in the vegetative life cycle is a collagenase-like enzyme which attacks only selected domains in its wall substrates containing (hydroxy)-proline clusters. Cell-wall fractions obtained by salt-extraction (NaClO₄) and oxidizing agents (NaClO₂) and the insoluble residue were tested as substrates. The most-crosslinked insoluble inner part of the wall is the best substrate for the sporangia autolysin. Oligosaccharides obtained from the insoluble cell-wall fraction of sporangia by hydrolysis with Ba(OH)₂ inhibit autolysin action. We conclude that the oligosaccharide side chains of wall substrates are essential for forming the reactive enzyme-substrate complex.Abbreviations CSW chlorite-soluble cell-wall fraction - ICW insoluble cell-wall fraction - PSW salt-soluble fraction - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Observations on the Internal Structure of the Spermatozoid of Dictyota

The spermatozoid of Dictyota is shown to be structurally morelike a vegetative zoospore than was the case in Fucus. The mitochondria,golgi, overall shape of the nucleus, nuclear membrane, fat bodies,and miscellaneous vesicles are as in a zoospore; there is, however,a vestigial chromatophore without an eyespot and the ciliaryapparatus is specialized. In spite of the single flagellum thereare two basal bodies inside the cell, one of which is apparentlyvestigial, ending blindy in the cytoplasm; both the a similarinternal structure with nine fibres in the wall. A fibrous ‘root’,possibly homologous with the ‘proboscis’ of Fucus,arises near the outer side of the functional basal body; itis a band of about eight fibres passing through the cell withouttouching the nucleus but closely pressed to the inner facesof at least two mitochondria before ending in an unknown mannerat the cell surface. The internal structure of the flagellumis normal but the fact that the median strand is marked by arowspines has been used to demonstrate is a conclusive way thefacts of bilateral symmetry in the whole organ; Dictyota agreesexactly with Fucus and a previous error based on incompleteinformation has been corrected. The process of unwinding ofthe flagellum from the surface of the body after liberationfrom the antheridium is described and used to illustrate someunexpected properties of the surface membrane.     

Occurrence of wall-less cells during synchronous gametogenesis in Chlamydomonas reinhardtii

Synchronous gametogenesis in Chlamydomonas reinhardtii is accompaniedby a round of cell division. Some of the unmated gametes becomenaked at daughter cell liberation. A sporangial wall-lytic enzyme,which is excreted into the medium at zoospore liberation, actson the wall of the gametic cell. (Received August 28, 1980; )     

Occurrence of wall-less cells during synchronous gametogenesis in Chlamydomonas reinhardtii

Synchronous gametogenesis in Chlamydomonas reinhardtii is accompaniedby a round of cell division. Some of the unmated gametes becomenaked at daughter cell liberation. A sporangial wall-lytic enzyme,which is excreted into the medium at zoospore liberation, actson the wall of the gametic cell. (Received August 28, 1980; )     

Cell Cycle Control in Arabidopsis

Although the basic mechanism of cell cycle control is conservedamong eukaryotes, its regulation differs in each type of organism.Plants have unique developmental features that distinguish themfrom other eukaryotes. These include the absence of cell migration,the formation of organs throughout the entire life-span fromspecialized regions called meristems, and the potency of non-dividingcells to re-enter the cell cycle. The study of plant cell cyclecontrol genes is expected to contribute to the understandingof these unique developmental phenomena. The principal regulatorsof the eukaryotic cell cycle, the cyclin-dependent kinases (CDKs)and cyclins, are conserved in plants. This review focuses oncell cycle regulation in the plant Arabidopsis thaliana . Whileexpression of one Arabidopsis CDK gene, Cdc2aAt, was positivelycorrelated with the competence of cells to divide, expressionof a mitotic-like cyclin, cyc1At, was almost exclusively confinedto dividing cells. The expression of the Arabidopsis -type cyclinsappears to be an early stage in the response of plant cellsto external and internal stimuli. Arabidopsis thaliana (L.) Heynh.; cell cycle; CDK; cyclin; plant development; plant hormone     

Proximal tubular epithelial cells are generated by division of differentiated cells in the healthy kidney

We searched for evidence for a contribution of stem cells in growth of the proximal S3 segments of healthy rats. According to the stem cell model, stem cells are undifferentiated and slow cycling; the bulk of cycling cells are transit amplifying, rapidly cycling cells. We show the following. 1) By continuous application of a thymidine analog (ThA) for 7 days, S3 proximal epithelial cells in healthy kidneys display a high-cycling rate. 2) Slow-cycling cells, identified by lack of ThA uptake during 14 days of continuous ThA application up to death and by expression of the cell cycle protein Ki67 at death, have the same degree of differentiation as quiescent cells. 3) To detect rapidly cycling cells, rats were killed at various time points after injection of a ThA. Double immunofluorescence for ThA and a cell cycle marker was performed, with colocalization indicating successive divisions. During one week after division, daughter cells display a very low proliferation rate, indicating the absence of rapidly cycling cells. 4) Labeling with cyclin D1 showed that this low proliferation rate is due to cycle arrest. 5) More than 50% of the S3 cells entered the cell cycle 36 h after a potent proliferative stimulus (lead acetate injection). We conclude that generation of new cells in the proximal tubule relies on division of differentiated, normally slow-cycling cells. These may rapidly enter the cycle under an adequate stimulus. immunohistochemistry; cell cycle; proliferation; renal stem cells; proximal tubule; renal epithelial cells     

TNF-alpha is a mitogen in skeletal muscle

Emerging evidence suggests that tumor necrosis factor (TNF)- plays a role in muscle repair. To determine whether TNF- modulates satellite cell proliferation, the current study evaluated TNF- effects on DNA synthesis in primary myoblasts and on satellite cell activation in adult mouse muscle. Exposure to recombinant TNF- increased total DNA content in rat primary myoblasts dose-dependently over a 24-h period and increased the number of primary myoblasts incorporating 5-bromo-2'-deoxyuridine (BrdU) during a 30-min pulse labeling. Systemic injection of TNF- stimulated BrdU incorporation by satellite cells in muscles of adult mice, whereas no BrdU was incorporated by satellite cells in control mice. TNF- stimulated serum response factor (SRF) binding to the serum response element (SRE) present in the c-fos gene promoter and stimulated reporter gene expression controlled by the same element. Our data suggest that TNF- activates satellite cells to enter the cell cycle and accelerates G₁-to-S phase transition, and these actions may involve activation of early response genes via SRF. cytokine; cell cycle; satellite cells; serum response factor; c-fos     

Cell Cycle Timing of Replication of the Nuclear Gene Encoding Chloroplastic NADP-Specific Glutamate Dehydrogenase Isoenzymes in Chlorella sorokiniana

In synchronized Chlorella sorokiniana cells, the NH₄⁺ inducibleNADP-specific glutamate dehydrogenase enzyme (NADP-GDH) accumulatedin a linear manner throughout the first cell cycle. Early inthe following second cell cycle, an increase in its rate ofaccumulation occurred that was proportional to the increasein total cellular DNA in the previous cell cycle. In synchronizedbacterial cells, increases in rate of linear accumulation ofinducible enzymes coincide with the time of replication of theirstructural genes. To determine whether the rate change in NADPGDHaccumulation resulted from a delay in replication of its nuclearstructural gene (gdhN) in fully induced C. sorokiniana cells,the cell cycle timing of replication of this gene was comparedto that of another nuclear gene, nitrate reductase (nia), andof a chloroplast gene, ribulose bisphosphate carboxylase large-subunit(rbcL), in synchronized cells cultured in NH₄⁺ or NO₃^–(uninduced) medium. The gdhN and nia genes replicated withinthe period of nDNA synthesis and rbcL within the period of ctDNAsynthesis in cells growing in either nitrogen source. Therefore,the delayed rate change in enzyme accumulation results froma process that regulates expression of the gdhN gene after itsreplication. (Received July 16, 1994; Accepted November 28, 1994)     

Studies on the cell wall of Chlorella I. Quantitative changes in cell wall polysaccharides during the cell cycle of Chlorella ellipsoidea

1. 1. The cell wall of Chlorella ellipsoidea was fractionated intotwo components, alkali-soluble hemicellulose and alkali-insoluble"rigid wall". The former was composed of several neutral sugars,i.e. rhamnose, xylose, arabinose, mannose and galactose, andthe latter had glucosamine as a main constituent sugar.
2. 2.Quantitative changes in both hemicellulose and "rigid wall"contents during the cell cycle were followed using synchronouslygrown cells. The two cell wall components showed markedly differentchanges. Hemicellulose increased in proportion to the enlargementof the cell surface area in the growing phase, while the "rigidwall" remained almost constant in this phase. The "rigid wall"increased only in the reproduction phase—the time of autosporeformation.

(Received September 26, 1977; )     

Cell cycle-related changes in transient K(+) current density in the GH3 pituitary cell line

Our aim was to determinewhether the expression of K⁺ currents is related to thecell cycle in the excitable GH3 pituitary cell line. K⁺currents were studied by electrophysiology, and bromodeoxyuridine (BrdU) labeling was used to compare their expression in cells thereafter identified as being in the S or non-S phase of the cellcycle. We show that the peak density of the transient outward K⁺ current (I_to) was 33% lower incells in S phase (BrdU+) than in cells in other phases of the cellcycle (BrdU). The voltage-dependence of I_towas not modified. However, of the two kinetic components ofI_to inactivation, the characteristics of thefast component differed significantly between BrdU+ and BrdU cells.Recovery from inactivation of I_to showedbiexponential and monoexponential function in BrdU and BrdU+ cells,respectively. This suggests that the molecular basis of this currentvaries during the cell cycle. We further demonstrated that4-aminopyridine, which blocks I_to, inhibited GH3cell proliferation without altering the membrane potential. These datasuggest that I_to may play a role in GH3 cellproliferation processes.

     

Cell cycle-dependent expression of volume-activated chloride currents in nasopharyngeal carcinoma cells

Patch-clamping and cell imageanalysis techniques were used to study the expression of thevolume-activated Cl current,I_Cl(vol), and regulatory volume decrease (RVD)capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated aCl current with a linear conductance, negligibletime-dependent inactivation, and a reversal potential close to theCl equilibrium potential. The sequence of anionpermeability was I > Br > Cl > gluconate. The Cl channelblockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),and ATP inhibited I_Cl(vol). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by adouble chemical-block (thymidine and hydroxyurea) technique. Theexpression of I_Cl(vol) was cell cycle dependent,being high in G₁ phase, downregulated in S phase, butincreasing again in M phase. Hypotonic solution activated RVD, whichwas cell cycle dependent and inhibited by the Cl channelblockers NPPB, tamoxifen, and ATP. The expression of I_Cl(vol) was closely correlated with the RVDcapacity in the cell cycle, suggesting a functional relationship.Inhibition of I_Cl(vol) by NPPB (100 µM)arrested cells in G₀/G₁. The data also suggest that expression of I_Cl(vol) and RVD capacity areactively modulated during the cell cycle. The volume-activatedCl current associated with RVD may therefore play animportant role during the cell cycle progress.

     

SYNCHRONOUS CULTURE OF CHLORELLA: I. KINETIC ANALYSIS OF THE LIFE CYCLE OF CHLORELLA ELLIPSOIDEA AS AFFECTED BY CHANGES OF TEMPERATURE AND LIGHT INTENSITY

1. Using the technique of synchronous culture, investigationsweremade of the effects of temperature and light-intensityon cellularlife cycle of Chlorella ellipsoidea. Some improvementsin theculture technique for obtaining a good synchrony of algalgrowthwere described.
2. By following the changes of averagecell volume and cell numberoccurring during culturing, therates of the following processesof life cycle were determined:(i) "growth" (or the increasein cell mass) occurring from thestage of smaller cells (D_a)to the stage of ripened cell (L₃),(ii) "ripening" (or processofformation of "nuclear substances"as estimated from the averagenumber of daughter cells formedfrom single mother cell), and(iii) " maturing and division" which leads to the full maturationof mother cells (L-cells)and their division into separate daughtercells (D-cells).
3. "Growth"and "ripening" were found to be dependent in light,"maturingand division" light-independent. The time requiredfor "growth"and "ripening" (C) is dependent on temperaturebut independentof light intensity, the onset of "maturing anddivision" occurringat the same time (D) of culturing undervaried light intensities.The average cell volume at this stage(L₃),however, was foundto be markedly modified by light intensity;larger with highertemperatures (see Fig. 4).
4. Changes in incubation temperature(under the condition of saturatinglight intensities) were foundto affect the life cycle in thefollowing way: (i) The timeof onset of "maturing and division"(D), varies markedly withculturing temperature; earlier athigher temperatures, (ii)The average cell volume at this stagealso depends on temperature; smaller at higher temperatures.
5. The average number of daughtercells (n) emerging from singlemother cells, was found to beuninfluenced by culturing temperature;(4.0–4.1 underthe conditions of the present study). Itwas found that thedivision number n is remarkably varied bychanging the lightintensity in the "growth" and "ripening"phases; 2.0 at 1 kilolux,3.7 at 5 kilolux, 4.2 at saturatinglight intensities (10 and25 kilolux). This finding was explainedby assuming a light-dependentformation of "nuclear substances"during the "growth" and "ripening"phases, the quantity of thesubstances in the cell at L₃ stagedeterminig the division number.
6. The experimental data wereanalyzed reaction kinetically, therate constants and othercharacteristics of the reactions constitutingthe processesof life cycle were determined, and values forthe apparent activationenergy for each reaction were computed.The reactions were discussedwith special reference to theirrelationship with photosyntheticprocess was discussed.

(Received November 7, 1959; )