Studies on the cell cycle of Myxobacter AL-1

The properties of seven enzymes were studied in extracts from Myxobacter AL-1. The enzymes were isocitrate dehydrogenase (E.C. 1.1.1.42), succinate dehydrogenase (E.C. 1.3.99.1), alkaline phosphatase (E.C. 3.1.3.1), -glucosidase (E.C. 3.2.1.20), -glucosidase (E.C. 3.2.1.21), -galactosidase (E.C. 3.2.1.23), and N-acetyl-glucosaminidase (E.C. 3.2.1.30). Four of these enzymes: isocitrate dehydrogenase, -glucosidase, -glucosidase, and -galactosidase are cytosolic enzymes. Succinate dehydrogenase was found to be located on the cytoplasmic membrane system, whereas alkaline phosphatase and N-acetyl-glucosaminidase were considered as enzymes which bind the outer membranes resp. the cell wall. During the cell cycle, all enzymes have a pattern of discontinuous activity increase. Succinate dehydrogenase and isocitrate dehydrogenase exhibit a stepwise increase of activity, whereas the other enzymes follow the pattern of a peak enzyme.     

The bifunctional enzyme chitosanase-cellulase produced by the gram-negative microorganism Myxobacter sp. AL-1 is highly similar to Bacillus subtilis endoglucanases

The gram-negative bacterium Myxobacter sp. AL-1 produces chitosanase-cellulase activity that is maximally excreted during the stationary phase of growth. Carboxymethylcellulase zymogram analysis revealed that the enzymatic activity was correlated with two bands of 32 and 35 kDa. Ion-exchange-chromatography-enriched preparations of the 32-kDa enzyme were capable of degrading the cellulose fluorescent derivatives 4-methylumbelliferyl-β-d-cellobioside and 4-methylumbelliferyl-β-d-cellotrioside. These enzymatic preparations also showed a greater capacity at 70° C than at 42° C to degrade chitosan oligomers of a minimum size of six units. Conversely, the β-1,4 glucanolytic activity was more efficient at attacking carboxymethylcellulose and methylumbelliferyl-cellotrioside at 42° C than at 70° C. The 32-kDa enzyme was purified more than 800-fold to apparent homogeneity by a combination of ion-exchange and molecular-exclusion chromatography. Amino-terminal sequencing indicated that mature chitosanase-cellulase shares more than 70% identity with endocellulases produced by strains DLG, PAP115, and 168 of the gram-positive microorganism Bacillus subtilis. Received: 6 January 1997 / Accepted: 29 May 1997     

Induction of alpha-glucosidase and synthesis during the cell cycle of Myxobacter AL-1.

Alpha-Glucosidase of Myxobacter AL-1 was induced by the addition of maltose to a defined medium as the sole carbon source. This induction takes place during lag-phase conditions without concomitant growth; it seems not to be regulated by the mechanism of catabolite repression. Using the method of density labeling with deuterated amino acids and subsequent analysis by equilibrium density gradient centrifugation in metrizamide-2H2O gradients it could be shown that the activity increase of the enzyme during induction is the the result of a de novo synthesis of the enzyme protein. After a short pulse of induction with maltose, alpha-glucosidase exhibited a pattern of variation in enzyme activity during the cell cycle that was similar to the one observed after growth of the cells on maltose for several generations.     

Reversible blockage of the cell cycle in the meristem of Riella helicophylla (Bory et Mont.) Mont. by p-chlorophenoxyisobutyric acid (PCIB)

Treatment of gemmae of Riella helicophylla with PCIB results in reversible blockage of the meristematic cells in the G1-phase. At the same time the normal subdivision of the intercalary meristem into two lateral meristems disappears. PCIB is effective only when the plants are exposed to light.Abbreviations PCIB p-chlorophenoxyisobutyric acid - IAA indoleacetic acid Dedicated to Professor Peter Karlson on the occasion of his 60^th birthday     

Developmental cell cohesion in Stigmatella aurantiaca

Cells of the myxobacterium Stigmatella aurantiaca increased in cohesiveness during the aggregation stage of fruiting body formation. Cell cohesion was genus specific, as determined by suspending S. aurantiaca cells with other bacteria and measuring their inclusion in the cell clumps. Cohesion between aggregating cells became resistant to moderate shear stresses (vortex mixing) approximately at the aggregate stage. Cells harvested prior to the appearance of aggregates formed shear sensitive followed by shear resistant cohesions in cell suspension.     

On the structure of the peptidoglycan of cell walls from Myxobacter AL-1 (myxobacterales).

Basically the peptidoglycan of Myxobater AL-1 consists of alternating beta-1,4-linked N-acetylglucosamic-N-acetylmuramic acid chains. After splitting the aminosugar backbone with a specific algal enzyme three subunits arise: a monomer, a dimer and a timer. Investigation of the monomer with specific enzymes and comparison of the degradation products to standards derived from other bacterial peptidoglycans suggest the following structure of the monomer peptide: L-alanyl-D-glutamic-L-meso-diaminopimelic-D-alanine. A D-alanyl-D-meso-diaminopimelic acid bond is the bridgebond between the peptides of the subunits.     

On the structure of the peptidoglycan of cell walls from Myxobacter AL-1 (Myxobacterales)

Basically the peptidoglycan of Myxobater AL-1 consists of alternating β-1,4-linked N-acetylglucosamic-N-acetylmuramic acid chains. After splitting the aminosugar backbone with a specific algal enzyme three subunits arise: a monomer, a dimer and a trimer. Investigation of the monomer with specific enzymes and comparison of the degradation products to standards derived from other bacterial peptidoglycans suggest the following structure of the monomer peptide: l-alanyl-d-glutamic-l-meso-diaminopimelic-d-alanine. A d-alanyl-d-meso-diaminopimelic acid bond is the bridgebond between the peptides of the subunits.     

Prolyl oligopeptidase participates in cell cycle progression in a human neuroblastoma cell line

Prolyl oligopeptidase (POP) is a post-proline cleaving enzyme, which is widely distributed in various organs, with high levels in the brain. In this study, we investigated the effects of a selective POP inhibitor, 3-({4-[2-(E)-styrylphenoxy]butanoyl}-l-4-hydroxyprolyl)-thiazolidine (SUAM-14746), on the growth of NB-1 human neuroblastoma cells. SUAM-14746 treatment for 24–72 h suppresses the growth of NB-1 cells without cell death in a dose-dependent manner (10–60 μM). Similar suppressive effects were observed with another POP inhibitor benzyloxycarbonyl-thioprolyl-thioprolinal. The SUAM-14746-induced growth inhibition in NB-1 cells was associated with pronounced G₀/G₁ arrest and reduced levels of phosphorylated retinoblastoma protein (pRb), cyclin E, and cyclin dependent kinase (CDK) 2, and increased levels of the CDK inhibitor p27^kip1 and the tumor suppressor p53. SUAM-14746 also induced transient inhibition of S and G₂/M phase progression, which was correlated with retardation of the decrease in the levels of cyclins A and B. Moreover, RNAi-mediated knockdown of POP also led to inhibition of NB-1 cell growth and the effect was accompanied by G₀/G₁ arrest. These results indicate that POP is a part of the machinery that controls the cell cycle.     

Factors regulating the expression of cell cycle genes in individual buds ofPopulus

The early and differential responses of the individual buds along a shoot have remained largely unknown due to the difficulties of analyzing early indicators that allow the monitoring of the effects of subtle changes in the environment on the growth activity of the individual bud. To overcome this problem, we transformed poplar [Populus tremula (L.) xP. alba (L.)] with two chimeric genes,Pcdc2a-gus andPcycl At-gus, the expression of which is closely linked to cell division inArabidopsis thaliana (L.) Heynh. We analyzed the expression levels of both chimeric genes in individual buds of the same tree, and under different conditions known to promote or retard growth in the buds. The expression levels of both chimeric genes were found to reflect closely the growth activity of the buds. After decapitation of the shoot, the expression ofPcdc2a-gus andPcycl At-gus revealed rapid and selective changes in the cell cycle, even when no morphological changes were observed. Furthermore, on the basis of the expression of the chimeric cell cycle genes, different degrees of growth activity and dormancy could be discriminated in the axillary buds. In addition, the expression ofPcycl At-gus was found to be closely associated with the day length, which is critical for dormancy induction in poplar.Abbreviations GUS -glucuronidase - MU methylumbelliferone     

A flow fluorimetric analysis of the cell cycle during growth and differentiation inDictyostelium discoideum

Summary We report a flow fluorimetric analysis of the DNA content of cells and nuclei from vegetative populations and various developmental stages of the cellular slime mouldDictyostelium discoideum using the dyes Hoechst 33258 and mithramycin. Nuclei from all of these populations showed an identical single DNA-content peak, indicating that most vegetative cells and most cells in all developmental stages are in one phase of the cell cycle. Our own data and findings in the literature indicate that this phase is G₂. On the other hand, we also found that various stages, subpopulations of cells at early stages and the different differentiated cell types in the slug stage differ in DNA content per cell. Any particular population typically has one major peak of DNA content, with a modal value that is characteristic for the cell type and for the developmental stage. These differences presumably reflect differences in mitochondrial DNA content per cell.     

Changes in the rate of synthesis of wall polysaccharides during the cell cycle of yeast

Reevaluation and comparison of seemingly contradictory literature data on the mode of synthesis of wall polysaccharides during the cell cycle ofSaccharomyces cerevisiae explained the source of discrepancies and demonstrated their general consonance in the following points: 1. The rate of synthesis of glucan and mannan is not constant and does not increase continuously throughout the entire cell cycle. 2. The rate of synthesis of both polysaccharides is considerably reduced at the time of cell division and in the prebudding phase.     

Investigations on the cell cycle of haploid and diploid tissue cultures of Datura innoxia Mill. and its synchronization

Using a ¹⁴C/³H double-labelling technique, the influence of kinetic on the length of the cell cycle of meristematic cells in haploid and diploid callus cultures of Datura innoxia was determined. The total length of the cell cycle of haploid cells as compared to that of diploid cells was reduced by 2.3 h (-kinetin) or 1.4 h (+kinetin). Furthermore, the addition of kinetin to the nutrient solution also reduces cell cycle duration at both ploidy levels. For synchronization of the cell cycle, a fluorodesoxyuridine/thymidine system was successfully employed. Apparently, the reduction of total cell cycle duration of cycling cells due to treatment with kinetin occurred at the expense of the G₁phase. Nevertheless, kinetin seems to exert an influence on the transition of cells from the G₂ into the M phase as well.Abbreviations FUdR fluorodeoxyuridine - HU hydroxyurea - IAA nidole acetic acid     

Temperature response of the cell cycle of Haplopappus gracilis in suspension culture and its significance to the G1 transition probability model

The effects of temperature on the cell cycle of Haplopappus gracilis suspension cultures were analysed by the fraction of labelled mitoses method. Sphase in these cultures shows a different temperature optimum as compared to optima derived for G₂ and mitosis. G₁ phase has a much lower Q₁₀ than the other cell cycle phases and shows no temperature optimum between 22 and 34° C. These results are discussed in relation to a transition probability model of the cell cycle proposed by Smith and Martin (Proc. Natl. Acad. Sci. USA 70, 1263–1267, 1973), in which each cell has a time independent probability of initiating the transition into another round of DNA replication and division. The implications of such a model for cell cycle analysis are discussed and a tentative model for a probabilistic transition trigger is advanced.Abbreviations FLM Fraction of labelled mitoses - T_B Total B-phase     

Growth and development in relation to the cell cycle inPhysarum polycephalum

Summary In strain CL ofPhysarum polycephalum, multinucleate, haploid plasmodia form within clones of uninucleate, haploid amoebae. Analysis of plasmodium development, using time-lapse cinematography, shows that binucleate cells arise from uninucleate cells, by mitosis without cytokinesis. Either one or both daughter cells, from an apparently normal amoebal division, can enter an extended cell cycle (28.7 hours compared to the 11.8 hours for vegetative amoebae) that ends in the formation of a binucleate cell. This long cycle is accompanied by extra growth; cells that become binucleate are twice as big as amoebae at the time of mitosis. Nuclear size also increases during the extended cell cycle: flow cytometric analysis indicates that this is not associated with an increase over the haploid DNA content. During the extended cell cycle uninucleate cells lose the ability to transform into flagellated cells and also become irreversibly committed to plasmodium development. It is shown that commitment occurs a maximum of 13.5 hours before binucleate cell formation and that loss of ability to flagellate precedes commitment by 3–5 hours. Plasmodia develop from binucleate cells by cell fusions and synchronous mitoses without cytokinesis.Abbreviations CL Colonia Leicester - DSDM Dilute semi-defined medium - FKB Formalin killed bacterial suspension - IMT Intermitotic time - LIA Liver infusion agar - SBS Standard bacterial suspension - SDM Semi-defined medium     

Heme oxygenase-1 expression levels are cell cycle dependent

Heme oxygenase-1 (HO-1) is a stress protein, which has been suggested to participate in defense mechanisms against agents that may induce oxidative injury, such as angiotensin II (Ang II). The purpose of the present study was to examine the role of human HO-1 in cell-cycle progression. We investigated the effect of Ang II on HO-1 gene expression in serum-deprived media to drive human endothelial cells into G(0)/G(1) (1% FBS) compared to exponentially grown cells (10% FBS). The addition of Ang II (100 ng/ml) to endothelial cells increased HO-1 protein and activity in G(0)/G(1) in a time-dependent manner, reaching a maximum HO-1 level at 16 h. Real-time RT-PCR demonstrated that Ang II increased the levels of HO-1 mRNA in G(0)/G(1) as early as 1 h. The rate of HO-1 induction in response to Ang II was several-fold higher in serum-starved cells compared to cells cultured in continuous 10% FBS. The addition of Ang II increased the generation of 8-epi-isoprostane PGF(2 alpha). Inhibition of HO-1, by Stannis mesoporphyrin (SnMP), potentiated Ang II-mediated DNA damage and generation of 8-epi-isoprostane PGF(2 alpha). These results imply that expression of HO-1 in G(0)/G(1), in the presence of Ang II, may be a key player in attenuating DNA damage during cell-cycle progression. Thus, exposure of endothelial cells to Ang II causes a complex response involving generation of superoxide anion, which may be involved in DNA damage. Upregulation of HO-1 ensures the generation of bilirubin and carbon monoxide (CO) in G(0)/G(1) phase to counteract Ang II-mediated oxidative DNA damage. Inducibility of HO-1 in G(0)/G(1) phase is essential and probably regulated by a complex system involving oxygen species to assure controlled cell growth.     

Growth kinetics of the Golgi apparatus during the cell cycle in onion root meristems

In onion root meristems, the number of dictyosomes per cell shows a kinetics of growth strongly related to the cell cycle. During the interphase of steady-state proliferative cells, the volume density and numerical density of the Golgi apparatus decrease to reach minimum values in late-interphase cells, characterized by their greatest length. This pattern is also found in the total volume occupied by Golgi apparatus. Once in mitosis, the above-mentioned parameters begin to increase reaching maximum mean values in telophase. After the experimental uncoupling of chromosome and growth cycles by presynchronization with hydroxyurea, we found a similar behaviour pattern in the Golgi apparatus: decreasing values during interphase and a triggering of Golgi-apparatus growth in prophase independently of the bigger cell sizes reached in mitosis as an effect of pretreatment with hydroxyurea. These results indicate a cyclic kinetics of this subcellular component in higher-plant meristems, coupled with early mitotic events.     

Studies on ultrastructure and composition of cell walls of the cyanobacterium Anacystis nidulans

The multilayered cell wall of the cyanobacterium Anacystis nidulans was studied by the freezeetching technique. A characteristic fracture face in the outer cell wall was demonstrated which is densely packed with particles of a diameter of 60–75 Å. This particle layer is comparable with layers which have been described in many cell walls of Gram-negative prokaryotes.The outer membrane of the cell wall was solubilised by extraction with phenol/water or sodium dodecyl sulfate (SDS). In the SDS-extract 31 bands were separated by polyacrylamide gel electrophoresis, among them 3–5 major proteins with molecular weights of approximately 60, 40, and 10 kdaltons, respectively. Several polypeptides of the Anacystis cell wall were comparable in their mobility with polypeptides extracted from cell walls of different Gramnegative bacteria. The analysis of the SDS-unsoluble electron dense layer (sacculi) revealed the typical components of peptidoglycan diaminopimelic acid, muramic acid, glutamic acid, glucosamine and alamine in the molar ratio of 1.0:0.9:1.1:1.5:1.9. In addition, other amino acids (molar ratio from 0.05–0.36), mannosamine (molar ratio 0.54), and lipopolysaccharide components were detected in low concentration.Abbreviations SDS sodium dodecyl sulfate - EDTA ethylene diamine tetraacetate     

TheCaesalpinia peltophoroides cell cycle under different aeration conditions

Summary Cell cycle parameters were studied inCaesalpinia peltophoroides meristems proliferating under different oxygen tensions. This species has been selected for mixed planting in degraded areas in Brazil, some of which are occasionally flooded. As the species’ adaptation to oxygen deprivation during flooding is not fully understood, the objective of this study was to characterize the meristematic activity of root cells under various oxygen regimes. Synchronous binucleate cells, induced by a pulse of caffeine, showed a cell-cycle time constant under both control (5.6 mg of O₂ per l) and oxygenated conditions (7.9 and 3.2 mg of O₂ per l). The whole cell cycle lasted 10 h, although the relative duration of metaphase and anaphase/early telophase increased in more hypoxic conditions. The species appeared to utilise oxygen diffusing from the shoot to the root system to maintain cell division and root growth.     

Effect of abscisic acid on the cell cycle in the growing maize root

The mechanism by which the rate of cell proliferation is regulated in different regions of the root apical meristem is unknown. The cell populations comprising the root cap and meristem cycle at different rates, proliferation being particularly slow in the quiescent centre. In an attempt to detect the control points in the cell cycle of the root apical meristem of Zea mays L. (cv. LG 11), quiescent-centre cells were stimulated to synthesise DNA and to enter mitosis either by decapping or by immersing intact roots in an aqueous 3,3-dimethyl-glutaric acid buffer solution. From microdensitometric and flow-cytometric data, we conclude that, upon immersion, the G₂ phase of the cell cycle of intact roots was shortened. However, when 50 M abscisic acid (ABA) was added to the immersion buffer, parameters of the cell cycle were restored to those characteristic of intact roots held in a moist atmosphere. On the other hand, decapping of primary roots preferentially shortened the G₁ phase of the cell cycle in the quiescent centre. When supplied to decapped roots, ABA reversed this effect. Therefore, in our model, applied ABA retarded the completion of the cell cycle and acted upon the exit from either the G₁ or the G₂ phase. Immersion of roots in buffer alone seems to trigger cells to more rapid cycling and may do so by depleting the root of some ABA-like factor.Abbreviations ABA cis-abscisic acid - DGA 3,3-dimethyl-glutaric acid - DAPI 4,6-diamidino-2-phenylindole - LI labelling index We thank Pierre Zaech of the Ludwig Institute, Epalinges, Switzerland, for expert assistance in flow cytometry and Dr. Jean-Marcel Ribaut of our Institute for providing data on exodiffusion and metabolism of ABA.