Synchronous Cultures Research Articles

The role of nucleotide binding and hydrolysis in the function of the fission yeast cdc18(+) gene product.

The fission yeast cdc18(+) gene is required for both initiation of DNA replication and the mitotic checkpoint that normally inhibits mitosis in the absence of DNA replication. The cdc18(+) gene product contains conserved Walker A and B box motifs. Studies of other ATPases have shown that these motifs are required for nucleotide binding and hydrolysis, respectively. We have observed that mutant strains in which either of these motifs is disrupted are inviable. The effects of these mutations were examined by determining the phenotypes of mutant strains following depletion of complementing wild-type Cdc18. In both synchronous and asynchronous cultures, the nucleotide-hydrolysis motif mutant (DE286AA) arrests with a 1C-2C DNA content, and thus exhibits no obvious defects in entry into S phase or in the mitotic checkpoint. In contrast, in cultures synchronized by hydroxyurea arrest and release, the nucleotide-binding motif mutant (K205A) exhibits the null phenotype, with 1C and <1C DNA content, indicating a block in entry into S phase and loss of checkpoint control. In asynchronous cultures this mutant exhibits a mixed phenotype: a percentage of the population displays the null phenotype, while the remaining fraction arrests with a 2C DNA content. Thus, the phenotype exhibited by the K205A mutant is dependent on the cell-cycle position at which wild-type Cdc18 is depleted. These data indicate that both nucleotide binding and hydrolysis are required for Cdc18 function. In addition, the difference in the phenotypes exhibited by the nucleotide-binding and hydrolysis motif mutants is consistent with a two-step model for Cdc18 function in which nucleotide binding and hydrolysis are required for distinct aspects of Cdc18 function that may be executed at different points in the cell cycle.

Changes in ascorbate oxidase gene expression and ascorbate levels in cell division and cell elongation in tobacco cells

The biological function of ascorbate oxidase (AAO) was not yet clarified, although it was suggested that AAO may be involved in cell growth. We investigated AAO expression and ascorbate metabolism during non‐synchronous, synchronous, and elongation cultures of tobacco BY‐2 cells. In non‐synchronous culture, AAO mRNA was abundant in logarithmic growth phase. Ascorbate content greatly increased during the growth, whereas dehydroascorbate content was slightly increased. In synchronous division culture, AAO mRNA was detected in all phases, but the levels were quite low in G1 phase. Ascorbate content was high in all phases, whereas dehydroascorbate content was low, especially in G1 phase. In elongation culture, the levels of AAO mRNA increased during elongation of the cells. AAO activity in the culture medium, as well as ascorbate and dehydroascorbate contents in the cells, also increased during the elongation. We propose that AAO expression and production of dehydroascorbate are under the control of the cell cycle and that AAO may function apoplastically as an ascorbate oxidizer in the process of cell elongation.

Different constitutive heat shock protein 70 expression during proliferation and differentiation of rat C6 glioma cells.

Analysis of constitutive heat shock protein 70 (HSC70) concentration in unstressed proliferating and differentiated rat C6 glioma cells revealed a striking reduction in the amount of HSC70 in differentiated cells. Proliferating cells showed a significantly higher HSC70 concentration, particularly observable during S phase in synchronous cultures. The activity of the cAMP/PKA signaling pathway was enhanced in differentiated cells. cAMP-elevating treatments both inhibited growth and reduced HSC70 concentration. Inactivation of PKA by H-89 upregulated the reduced HSC70 expression in differentiated cells and stimulated proliferation. Treatment with an inhibitor of MAP kinase activation (PD98059) reduced the HSC70 concentration. We assume that cAMP does not directly inhibit HSC70 expression by transcriptional repression, but by its inhibitory effect on the MAP kinase pathway.

Synchronization of yeast cell populations.

The study of synchronous populations of yeast cells has provided a wealth of information into regulatory aspects of the eukaryotic cell division cycle. Synchronized yeast cultures may also have potential benefit when exploiting yeasts in biotechnology. This paper provides an overview of the methods which have been used in the synchronization of cell division in budding and fission yeasts. The relative merits of these methods are outlined and protocols for preferred synchronization methods, based on size selection techniques, are described. In particular, centrifugal elutriation protocols for Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast) are detailed as this method is regarded as one of the best ways of preparing 'unperturbed' synchronous yeast cultures for cell cycle studies.

Orderly formation of the double ring structures for plastid and mitochondrial division in the unicellular red alga Cyanidioschyzon merolae

The formation of the plastid-dividing ring (PD ring) and mitochondrion-dividing ring (MD ring) was studied in a highly synchronous culture of the unicellular red alga Cyanidioschyzon merolae. The timing and the order of formation of the MD and PD rings were determined by observing organelles around the onset of their division, using transmission electron microscopy. In C. merolae, there is one chloroplast and one mitochondrion per cell, and the shape of the chloroplast changes sequentially from acorn-like, to round, to trapezoidal, to peanut-shaped, in that order, during the early stage of chloroplast division. None of the cells with acorn-shaped or round chloroplasts contained organelles with PD rings or MD rings, while all of the cells with peanut-shaped chloroplasts contained organelles with both PD rings and MD rings. In cells with peanut-shaped chloroplasts, the PD and MD rings were double ring structures, with an outer ring located on the cytoplasmic face of the outer membrane of the organelle, and an inner ring located in the matrix beneath the inner membrane. These results suggested that the double ring structures of the PD ring and the MD ring form when chloroplasts are trapezoidal in shape. Detailed three-dimensional observation of cells with trapezoidal chloroplasts revealed the following steps in the formation of the double ring structures of the PD and MD rings: (i) the inner ring of the PD ring forms first, followed by the outer ring; (ii) then the MD ring forms and becomes visible; (iii) when the double ring structures of the two rings have formed, the microbody then moves from its remote location to the plane of division of the mitochondrion and contraction of the PD and MD rings commences. These steps were also confirmed by computer-aided three-dimensional reconstruction of the images from serial thin sections. This study reveals the order of formation of the double ring structures of the PD and MD rings, and the behavior of the microbody around the onset of division of plastids and mitochondria. The results also provide the first evidence that the inner PD ring is not a tension element formed by the contractile pressure but a definite structure, independent of the outer ring.

Failure on induction of gametocytogenesis in synchronous culture of plasmodium falciparum

Failure on induction of gametocytogenesis in synchronous culture of plasmodium falciparum

A role for NIMA in the nuclear localization of cyclin B in Aspergillus nidulans.

NIMA promotes entry into mitosis in late G2 by some mechanism that is after activation of the Aspergillus nidulans G2 cyclin-dependent kinase, NIMXCDC2/NIMECyclin B. Here we present two independent lines of evidence which indicate that this mechanism involves control of NIMXCDC2/NIMECyclin B localization. First, we found that NIMECyclin B localized to the nucleus and the nucleus-associated organelle, the spindle pole body, in a NIMA-dependent manner. Analysis of cells from asynchronous cultures, synchronous cultures, and cultures arrested in S or G2 showed that NIMECyclin B was predominantly nuclear during interphase, with maximal nuclear accumulation in late G2. NIMXCDC2 colocalized with NIMECyclin B in G2 cells. Although inactivation of NIMA using either the nimA1 or nimA5 temperature-sensitive mutations blocked cells in G2, NIMXCDC2/NIMECyclin B localization was predominantly cytoplasmic rather than nuclear. Second, we found that nimA interacts genetically with sonA, which is a homologue of the yeast nucleocytoplasmic transporter GLE2/RAE1. Mutations in sonA were identified as allele-specific suppressors of nimA1. The sonA1 suppressor alleviated the nuclear division and NIMECyclin B localization defects of nimA1 cells without markedly increasing NIMXCDC2 or NIMA kinase activity. These results indicate that NIMA promotes the nuclear localization of the NIMXCDC2/ NIMECyclin B complex, by a process involving SONA. This mechanism may be involved in coordinating the functions of NIMXCDC2 and NIMA in the regulation of mitosis.

Selective synchronization of Tetrahymena pyriformis cell populations and cell growth kinetics during the cell cycle.

A selection synchronization technique based on ingestion of tantalum particles has been applied to obtain synchronized cultures of the filter feeding ciliate Tetrahymena pyriformis. Cell concentrations and cell volume distributions of synchronized cell populations have been monitored for more than four average generation times. A simple curve-fitting method has been used to decompose the cell volume distributions of a synchronous population into two populations representing cells before and after cell division. In this way, the time course of the growth of an initially synchronous culture is decomposed into the growth of successive generations. The data indicate that at any given time only two generations of cells are present in significant numbers. The measured cell volume distributions show that T. pyriformis has a complicated growth pattern during the cell cycle. Newborn T. pyriformis cells do not grow significantly at the beginning of the cell cycle. After the nongrowing stage, cells start growing in a possibly exponential rate before cells enter into a second nongrowing stage. The second nongrowing stage lasts until cell division. The presented data demonstrate that growing cell populations can be viewed as the composite of cells belonging to different generations. This concept has important implications for solving corpuscular models of cell growth.

Kim nasmyth: the universal truth

Kim nasmyth: the universal truth

In vitro growth inhibition of Plasmodium falciparum by retinol at concentrations present in normal human serum

To assess the in vitro effect of retinol on Plasmodium falciparum, the standard isolates 3D7, D10, W2 and K1 in continuous culture were exposed to retinol added in concentrations ranging from 10 −7 to 0.1 μmol/l. Parasite growth inhibition was assessed from 3H-hypoxanthine incorporation. Triplicate experiments were performed at physiological pH and in the case of D10, additional experiments were performed at pH 7.2 and 7.6. Final media retinol concentrations were assayed using high performance liquid chromatography. Retinol inhibited growth of both asynchronous and synchronous cultures of 3D7 and D10 and asynchronous cultures of W2 and K1. IC 50 values determined from assayed media concentrations ranged from 0.2 to 3.9 μmol/l and were comparable to concentrations in normal human serum (1.0–3.0 μmol/l). IC 50 values for asychronous D10 cultures at pH 7.2 were lower than at pH 7.4 or 7.6 (0.5, 3.9 and 5.0 μmol/l, respectively); results from synchronous cultures were similar. These data suggest that P. falciparum is a retinol-sensitive parasite, especially at pH levels equivalent to those in an acidotic patient. Adjunctive retinol therapy may have a role in clinical management of malaria.

Delta-9 fatty acid desaturase gene containing a carboxyl-terminal cytochrome b5 domain from the red alga Cyanidioschyzon merolae

A delta-9 fatty acid desaturase gene, homologous to animal and fungal acyl-coenzyme A (CoA) desaturases, was isolated from the red alga Cyanidioschyzon merolae using a degenerate PCR strategy. This gene, designated as CmFAD9, has no intron. The encoded delta-9 fatty acid desaturase (CmFad9p) consists of 476 amino acids and has an estimated molecular mass of 55.4 kDa. CmFad9p is a unique delta-9 fatty acid desaturase among plants, in that it is fused with the cytochrome b5 domain at its carboxyl terminus. This is characteristic of yeast acyl-CoA desaturase. Genomic Southern hybridization suggested that the C. merolae genome contains a single gene for delta-9 fatty acid desaturase of the animal and fungal type. Southern hybridization combined with pulsed-field gel electrophoresis revealed that CmFAD9 is probably located on chromosome XI of the 17 C. merolae chromosomes. A 1.6-kb product of this gene was transcribed throughout a light/dark synchronization culture. The discovery of CmFAD9 indicates the existence of a novel type of plant delta-9 fatty acid desaturase that may function in the endoplasmic reticulum, but not in the plastid.

Diel periodicity of glutamine synthetase activity during the cell cycle of Emiliania huxleyi

Light-dark regulation of glutamine synthetase (GS; EC 6.3.1.2) was investigated by following the development of activity and the time-course of the cell-cycle of the marine coccolithophorid, Emiliania huxleyi, under a 16-h photoperiod in synchronous cultures. GS activity showed marked diel periodicity by exhibiting a maximum value during the dark period and a minimum during the light. Our results suggest that regulation of GS activity during the light-dark cycle probably does not operate only through activation/deactivation mechanisms. Cell division took place from the end of the dark to the first hours of the light period. The succession of the cell-cycle events was analysed by flow cytometry. Variations in GS activity appeared to match the demands of the cell for glutamine, increasing during the G 1 phase in the light, and remaining at a high level during DNA synthesis in the dark.

In vitro antimalarial activity of penduline, a bisbenzylisoquinoline from Isopyrum thalictroides.

Two bisbenzylisoquinolines, tetrandrine and penduline, were purified from Isopyrum thalictroides. When tested for antimalarial activity in vitro, penduline was efficient at concentrations fivefold lower than those of tetrandrine. In highly synchronized parasite cultures, penduline mostly interfered between the 8th and the 32nd hours of the parasite cycle.

Simulations of the predator-prey interactions in a two species ecotoxicological test system

A mathematical simulation model (Gutierrez type) has been developed to investigate the predator-prey interactions in a two species ecotoxicological test system. The test system consists of the predacious mite ( Hypoaspis aculeifer Canestrini) and, as prey, the collembolan Folsomia fimetaria L. in a microcosm (5.5 cm high × 6.0 cm in diameter) with 30 g humidified soil. F. fimetaria is provided with bakers yeast as food source. The model simulates (i) the population development of the test animals in synchronous cultures before the test, (ii) the selection of the test animals from the synchronous cultures and (iii) predator-prey interactions with and without the presence of dimethoate during the test and extraction periods. The driving variable is the temperature dependent food demands of the organisms. Both species were simulated with separate male and female populations, which were divided into a number of life stages. Thus the model handles very complicated predator-prey interactions taking the different interactions between different instars into consideration. The simulated test results were found to be very sensitive to changes in predator search rate, capture efficiency, habitat overlap, the amount of stored reserves of H. aculeifer at test start and the survival and reproduction of F. fimetaria. The simulations constitute the foundation for suggestions for further improvement of the test system.

Identification of a new cellular protein that can interact specifically with DAN.

Recently, we demonstrated that the DAN gene product contains a growth- and/or a tumor-suppressive activity in vitro. In the present work, using a yeast two-hybrid system, we searched for cellular proteins that can associate with the DAN gene product. A cDNA clone, termed DA41, was initially isolated from an adult rat lung cDNA library. The DA41 gene was expressed in all adult tissues examined, however, the levels of expression varied significantly among the different tissues. Like the DAN gene product, the DA41 protein was similarly restricted to the cytoplasm. Sequence analysis revealed that DA41 cDNA is 2,167 nucleotides in length and contains a single open reading frame (ORF) of 582 amino acids (61,945 daltons). A homology search revealed that the DA41 gene product shares no structural similarity with those filed in the data base. In a synchronous 3Y1 cell culture, DA41 mRNA was expressed at a low level in quiescent cells; however, its level was significantly increased between the G1 and S phases of the cell cycle. On the other hand, the expression level of DAN mRNA did not change throughout the cell cycle progression. These results suggest that the DAN-DA41 complex might play a crucial role in the regulation of the cell cycle progression.

GROWTH-CONTROLLED OSCILLATION IN ACTIVITY OF HISTONE H1 KINASE DURING THE CELL CYCLE OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Synchronous cultures of the cell wall-less mutant Chlamydomonas reinhardtii Dangeard cw 15 were grown under different mean irradiances and different illumination regimes, which produced cell cycles that differed in the number of daughter cells released from one mother cell, in the length of the cell cycle, and in the growth rate. During the cell cycle, the cells reached several commitment points whose number and timing differed according to the particular pattern of the cell cycle. The cell volume was used as a growth parameter and increased in a stepwise manner. Each of the steps consisted of periods of both fast and slow growth. Growth usually stopped when the cells attained a volume twice that of the preceding step. Reaching particular commitment points was coupled with the position of these points in the enlargement of cell volume. Changes in the activity of histone H1 kinase were noted during the cell cycles of all experimental variants, and the activities were compared with the timing of various commitment points. It was found that kinase activity varied markedly within a single cell cycle, attaining maximal values when the cellular volume had doubled. Each peak in kinase activity slightly preceded the commitment to an individual sequence of reproductive events. In addition to the oscillations related to cell growth, a peak of kinase activity always occurred toward the end of the cell cycle when multiple rounds of DNA replication, mitosis, and cell division occurred.

Thymineless death in colon carcinoma cells is mediated via fas signaling.

Fas is expressed constitutively in colonic epithelial cells and is also expressed in colon carcinomas and in cultured colon carcinoma cell lines. However, the potential role of Fas signaling in mediating apoptosis in cells of this type remains unknown. We have developed human colon carcinoma cell models deficient in thymidylate synthase that demonstrate acute (TS- cells) or delayed (Thy4 cells) apoptosis following DNA damage induced by thymineless stress. Complete protection of cells from acute apoptosis and prolongation of delayed apoptosis was obtained following exposure to the NOK-1 monoclonal antibody (inhibitory to Fas signaling) during the period of dThd deprivation. These results suggested that apoptosis induced by thymineless stress was regulated by autocrine signaling via Fas-FasL interactions. Fas expression was high in both TS- and Thy4 cells. However, FasL, undetectable in synchronous cultures, was up-regulated in TS- cells at 48 hr, when cells were undergoing acute apoptosis, and in Thy4 cells at 96 hr, correlating with the delayed onset of thymineless death. FasL expression also correlated with acute apoptosis induced in parental GC3/cl cells, commencing at 48 hr, following thymidylate synthase inhibition by 5-fluorouracil/leucovorin exposure. Fas-mediated apoptosis induced by the cytotoxic anti-Fas monoclonal antibody CH-11 was inhibited following adenoviral delivery of a Bcl-2 cDNA, and Bcl-2 also protected cells from acute apoptosis induced by dThd deprivation. Taken together, these data demonstrate a functional Fas system in these cultured colon carcinoma cell models, and they demonstrate that Fas-FasL interactions can link DNA damage induced by thymineless stress to the apoptotic machinery of colon carcinoma cells.

Cell cycle dependency of rice α-amylase production in a recombinant yeast

The cell cycle dependency of rice alpha-amylase production in Saccharomyces cerevisiae was investigated using synchronous and arrested cultures. The results of two separate synchronous cultures, using alpha-mating factor and a cdc28 mutant, indicated that the rice alpha-amylase-specific production rate is not constant during the cell cycle. The specific production rates during G1, S, and M phases were then ascertained by inhibiting the progression of the cell cycle using alpha-mating factor, hydroxyurea, and nocodazole, respectively. The specific production rate was found to be maximal during the M phase. The increase in the specific production rate during the M phase was confirmed from the accumulation of M-phase cells using a cdc15 mutant. The intracellular content of rice alpha-amylase was also measured during the cell cycle. Like the specific production rate, the intracellular content was found to fluctuate throughout the cell cycle, and to reach a maximum during M phase.

Cell-cycle-regulated transcription of A- and B-type plant cyclin genes in synchronous cultures.

Synchronously dividing cell cultures of Catharanthus roseus were used to isolate cDNAs for two mitotic cyclins, named CYS and CYM. The deduced protein sequence of CYS is similar to that of A-type cyclins, and CYM belongs to the group of B-type cyclins. In a fashion similar to the pattern of expression seen for A-type and B-type cyclins in mammalian cells, CYS is expressed before CYM in C. roseus cells during the cell cycle. CYS mRNA accumulated at the onset of S phase and disappeared early in the G2 phase, whereas CYM mRNA was detected in the G2 and M phases of the cell cycle. Tobacco homologs of the two genes showed similar cell-cycle dependent expression patterns in synchronous cultures of tobacco BY2 cells. In both systems, CYS was expressed much earlier in the cell cycle than most other plant A-type cyclins, and hence CYS along with the soybean cyc1GM can be classified into a distinct subclass. The activities of CYM and CYS promoters during the cell cycle were analyzed in stably transformed tobacco BY2 cells. Cyclin promoter sequences of 0.5 kb could confer the typical cell-cycle-dependent expression to the beta-glucuronidase (GUS) reporter gene: the CYS promoter directed S-phase-specific expression, whereas the CYM promoter drove M-phase-specific expression. These results indicate the important role of transcriptional regulation in the oscillations of cyclin mRNA levels during the cell cycle.

Replication synchrony-PCR: a sampling-time-independent assay for replication synchrony in human tissues and tumors in situ.

Replication synchrony within a cell population can be demonstrated by pulse-labeling followed by PCR amplification of immunoprecipitated 5-iodo-2'-deoxyuridine (IdUrd)-labeled DNA from cells of otherwise indeterminant kinetic stages. This replication synchrony-PCR approach may be valuable in understanding the dynamics of human normal tissue or solid tumor replication in situ where access for repeated sampling is severely limited. IdUrd labeling provides a sampling-time-independent method for assessing the replicative status of a cell population at the time when the label was presented. Using genes whose time of replication in S phase is already known, the presence of a cell in early or late S phase can be determined and a qualitative measure made of replication synchrony in the population. This approach was evaluated in synchronous and random cultures of Ej cells using the early replicating PGK-1 gene to identify cells in early S phase at the time of labeling and the late replicating factor IX gene to identify cells that were in late S phase. To test the feasibility of clinical application of this technique, human tumor cells from patients with advanced cancers, given IdUrd therapeutically at specified times of the day, were evaluated. In some patients, replication synchrony-PCR provided evidence of parasynchronous DNA replication in tumor cells. This technique could be appended to existing clinical studies in which BrdUrd or IdUrd is being given to patients either diagnostically or therapeutically.