Synchronous Cultures Research Articles

DNA intermediates of meiotic recombination in synchronous S. pombe at optimal temperature.

Crossovers formed by recombination between homologous chromosomes are important for proper homolog segregation during meiosis and for generation of genetic diversity. Optimal molecular analysis of DNA intermediates of recombination requires synchronous cultures. We previously described a mutant, pat1-as2, of the fission yeast Schizosaccharomyces pombe that undergoes synchronous meiosis at 25°C when an ATP analog is added to the culture. Here, we compare recombination intermediates in pat1-as2 at 25°C with those in the widely used pat1-114 temperature-sensitive mutant at 34°C, a temperature higher than optimal. DNA double-strand breaks at most hotspots are similarly abundant in the two conditions but, remarkably, a few hotspots are distinctly deficient at 25°C. In both conditions, Holliday junctions at DNA break hotspots form more frequently between sister chromatids than between homologs, but a novel species, perhaps arising from invasion by only one end of broken DNA, is more readily observed at 25°C. Our results confirm the validity of previous assays of recombination intermediates in S. pombe and provide new information on the mechanism of meiotic recombination.

Plasmodium falciparum: Generation of pure gametocyte culture by heparin treatment

In vitro culture of Plasmodium falciparum gametocytes is essential for studying sexual development of the parasite. Here we describe a simple method for producing synchronous gametocyte culture without contamination of asexual stages. This method employs heparin’s activity in blocking merozoite invasion of erythrocytes to eliminate asexual stage parasites from gametocyte culture. We show that following induction of gametocyte formation, addition of heparin in culture medium for four days effectively eliminates asexual stages and produces pure, synchronous cultures of gametocytes. Compared with the commonly used N-acetylglucosamine treatment method, heparin treatment requires shorter time to eliminate asexual stages and causes significantly less hemolysis in late stage gametocyte cultures.

Characterization of the decline and recovery of heat-treated Scenedesmus vacuolatus.

BackgroundTo find out how algal cells cope with and recover from heat stress, the small vegetative cells of the synchronous Scenedesmus vacuolatus culture were subjected to a heat pretreatment (46.5°C for 1 h) followed by dark recultivation. The changes in physiological activities and morphology of Scenedesmus cells were continuously monitored throughout the course of decline and recovery.ResultsIt was found that the heat treatment, though completely inhibited photosynthesis, did not kill Scenedesmus cells. These cells, during dark recultivation, could make a fast repair and regained the ability of proliferation. We suggest that they entered a ‘stand-by’ state, which was characterized by condensed chromatin, partially functional but morphologically altered chloroplasts, disappeared vacuoles, slightly shrunk protoplast and intact plasma membranes. These stressed cells, on the surface, seemingly were undergoing some kind of disintegration, could readily and quickly return to normal cells upon illumination. Cell death occurred only after a long period of darkness (>48 h).ConclusionsOur results suggest that the recovery of algal cells from stress damage may actually proceed in two steps. The middle “stand-by’ stage normally is gone through too rapidly to be detected unless cells are kept in the dark.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-3) contains supplementary material, which is available to authorized users.

Cell dualism: presence of cells with alternative membrane potentials in growing populations of bacteria and yeasts

It is considered that all growing cells, for exception of acidophilic bacteria, have negatively charged inside cytoplasmic membrane (Δψ⁻-cells). Here we show that growing populations of microbial cells contain a small portion of cells with positively charged inside cytoplasmic membrane (Δψ⁺-cells). These cells were detected after simultaneous application of the fluorescent probes for positive membrane potential (anionic dye DIBAC⁻) and membrane integrity (propidium iodide, PI). We found in exponentially growing cell populations of Escherichia coli and Saccharomyces cerevisiae that the content of live Δψ⁻-cells was 93.6 ± 1.8 % for bacteria and 90.4 ± 4.0 % for yeasts and the content of live Δψ⁺-cells was 0.9 ± 0.3 % for bacteria and 2.4 ± 0.7 % for yeasts. Hypothetically, existence of Δψ⁺-cells could be due to short-term, about 1 min for bacteria and 5 min for yeasts, change of membrane potential from negative to positive value during the cell cycle. This change has been shown by the reversions of K⁺, Na⁺, and Ca²⁺ ions fluxes across the cell membrane during synchronous yeast culture. The transformation of Δψ(⁻-cells to Δψ⁺-cells can be explained by slow influx of K⁺ ions into Δψ⁻-cell to the trigger level of K⁺ concentration ("compression of potassium spring"), which is forming "alternative" Δψ⁺-cell for a short period, following with fast efflux of K⁺ ions out of Δψ⁺-cell ("release of potassium spring") returning cell to normal Δψ⁻ state. We anticipate our results to be a starting point to reveal the biological role of cell dualism in form of Δψ⁻- and Δψ⁺- cells.

Expression of p110, phosphorylation-protein kinase B and protein kinase B in hemangioma vascular endothelial cell in vitro

Objective To study the expression of phosphatidy linositol-3-kinase(PI3K) p110 catalytic subunit and phosphorylation-protein kinase B(p-Akt), protein kinase B(Akt) in cultured vascular endothelial cells, then the level of PI3K p110, p-Akt and Akt expression and the correlation with hemangioma vascular endothelial cell cycle and apoptosis were analyzed. Methods The hemangioma vascular endothelial cells which came from the sterile hemangioma were cultured by using tissue culture method and then synchronized cultured endothelial cells, PI3K p110, p-Akt and Akt of them were detected by way of Western blot, meanwhile cell cycle and cell apoptosis were examined by using flow cytometry technique, then the level of PI3K p110, p-Akt and Akt expression and the correlation with hemangioma vascular endothelial cell cycle and apoptosis were analyzed. Results The percentage of vascular endothelial cell was (94.00±3.00)% in the G0/G1 phase and the apoptosis rate was (11.24±2.34)% when p110 and p-Akt protein expression level were lower (0.19±0.01, 0.09±0.02), and they were (61.00±6.00)% and (0.51±0.03)% when p110 and p-Akt protein expression level were higher (0.37±0.04, 0.22±0.01). The percentage of vascular endothelial cell in the G0/G1 phase and the apoptosis rate were different in 2 groups(t=-8.42, -35.48, all P<0.01). But Akt protein expression level was not changed(0.72±0.00). Conclusions p110, p-Akt and Akt protein were expressed in the cultured hemangioma vascular endothelial cells in vitro.PI3K p110, p-Akt may be involved in cultured hemangioma vascular endothelial cells apoptosis in vitro. Key words: Hemangioma; Vascular endothelial cell; Cell culture; p110; Phosphorylation-protein kinase B; Protein kinase B; Cell apoptosis

Isolation and Classification of Bdellovibrio and Like Organisms

AbstractBdellovibrio and like organisms (BALOs) are obligate predators of Gram‐negative bacteria. BALOs are isolated as plaques growing at the expense of their prey and are cultivated as two‐member cultures. The growth cycle is composed of an extracellular attack phase and an intraperiplasmic elongation and replication phase. However, there are methods for obtaining host‐independent (HI) mutants that grow without prey on rich media. BALOs are commonly found in the environment but generally constitute small populations; therefore, their isolation may require enrichment steps. Contamination by other bacteria during isolation necessitates efficient separation between the smaller BALO cells from the majority of larger bacteria. BALOs can also be directly detected and quantified in environmental samples using specific PCR. Synchronous cultures of both wild‐type and HI derivatives can be obtained to study the different growth phases. These can be further separated by centrifugation. Classification is based on 16S rDNA analysis. Protocols relevant to these aspects of BALO detection, isolation, growth, classification, and quantitation are presented in this unit. Curr. Protoc. Microbiol. 26:7B.1.1‐7B.1.20. © 2012 by John Wiley &amp; Sons, Inc.

Preferential Fas-mediated apoptotic execution at G1 phase: the resistance of mitotic cells to the cell death

Apoptosis is induced by various stresses generated from the extracellular and intracellular environments. The fidelity of the cell cycle is monitored by surveillance mechanisms that arrest its further progression if any crucial process has not been completed or damages are sustained, and then the cells with problems undergo apoptosis. Although the molecular mechanisms involved in the regulation of the cell cycle and that of apoptosis have been elucidated, the links between them are not clear, especially that between cell cycle and death receptor-mediated apoptosis. By using the HeLa.S-Fucci (fluorescent ubiquitination-based cell cycle indicator) cells, we investigated the relationship between the cell cycle progression and apoptotic execution. To monitor apoptotic execution during cell cycle progression, we observed the cells after induction of apoptosis with time-lapse fluorescent microscopy. About 70% of Fas-mediated apoptotic cells were present at G1 phase and about 20% of cells died immediately after cytokinesis, whereas more than 60% of etoposide-induced apoptotic cells were at S/G2 phases in random culture of the cells. These results were confirmed by using synchronized culture of the cells. Furthermore, mitotic cells showed the resistance to Fas-mediated apoptosis. In conclusion, these findings suggest that apoptotic execution is dependent on cell cycle phase and Fas-mediated apoptosis preferentially occurs at G1 phase.

ATP analog-sensitive Pat1 protein kinase for synchronous fission yeast meiosis at physiological temperature

To study meiosis, synchronous cultures are often indispensable, especially for physical analyses of DNA and proteins. A temperature-sensitive allele of the Pat1 protein kinase (pat1-114) has been widely used to induce synchronous meiosis in the fission yeast Schizosaccharomyces pombe, but pat1-114-induced meiosis differs from wild-type meiosis, and some of these abnormalities might be due to higher temperature needed to inactivate the Pat1 kinase. Here, we report an ATP analog-sensitive allele of Pat1 [Pat1(L95A), designated pat1-as2] that can be used to generate synchronous meiotic cultures at physiological temperature. In pat1-as2 meiosis, chromosomes segregate with higher fidelity, and spore viability is higher than in pat1-114 meiosis, although recombination is lower by a factor of 2–3 in these mutants than in starvation-induced pat1+ meiosis. Addition of the mat-Pc gene improved chromosome segregation and spore viability to nearly the level of starvation-induced meiosis. We conclude that pat1-as2 mat-Pc cells offer synchronous meiosis with most tested properties similar to those of wild-type meiosis.

IFT Proteins Accumulate during Cell Division and Localize to the Cleavage Furrow in Chlamydomonas

Intraflagellar transport (IFT) proteins are well established as conserved mediators of flagellum/cilium assembly and disassembly. However, data has begun to accumulate in support of IFT protein involvement in other processes elsewhere in the cell. Here, we used synchronous cultures of Chlamydomonas to investigate the temporal patterns of accumulation and localization of IFT proteins during the cell cycle. Their mRNAs showed periodic expression that peaked during S and M phase (S/M). Unlike most proteins that are synthesized continuously during G1 phase, IFT27 and IFT46 levels were found to increase only during S/M phase. During cell division, IFT27, IFT46, IFT72, and IFT139 re-localized from the flagella and basal bodies to the cleavage furrow. IFT27 was further shown to be associated with membrane vesicles in this region. This localization pattern suggests a role for IFT in cell division.

Organic carbon source in formulated sediments influences life traits and gene expression of Caenorhabditis elegans

River water quality is strongly influenced by their sediments and their associated pollutants. To assess the toxic potential of sediments, sediment toxicity tests require reliable control sediments, potentially including formulated control sediments as one major option. Although some standardization has been carried out, one critical issue still remains the quality of sediment organic matter (SOM). Organic carbon not only binds hydrophobic contaminants, but may be a source of mild toxicity, even if the SOM is essentially uncontaminated. We tested two different sources of organic carbon and the mixture of both (Sphagnum peat (P) and one commercial humic substances preparation-HuminFeed(®), HF) in terms of life trait variables and expression profiles of selected life performance and stress genes of the nematode Caenorhabditis elegans. In synchronous cultures, gene expression profiling was done after 6 and 48 h, respectively. The uncontaminated Sphagnum P reduced growth, but increased numbers of offspring, whereas HF did not significantly alter life trait variables. The 6 h expression profile showed most of the studied stress genes repressed, except for slight to strong induction in cyp-35B1 (all exposures), gst-38 (only mixture), and small hsp-16 genes (all exposures). After 48 h, the expression of almost all studied genes increased, particularly genes coding for antioxidative defense, multiple xenobiotic resistance, vitellogenin-like proteins, and genes regulating lifespan. Overall, even essentially uncontaminated SOM may induce several modes of action on the molecular level in C. elegans which may lead to false results if testing synthetic xenobiotics. This contribution is a plea for a strict standardization of the SOM quality in formulated sediments and to check for corresponding effects in other model sediment organisms, especially if using molecular toxicity endpoints.

Ribozyme Stability, Exon Skipping, and a Potential Role for RNA Helicase in Group I Intron Splicing by Coxiella burnetii

The 23S rRNA gene of Coxiella burnetii, the agent of Q fever in humans, contains an unusually high number of conserved, selfish genetic elements, including two group I introns, termed Cbu.L1917 (L1917) and Cbu.L1951 (L1951). To better understand the role that introns play in Coxiella's biology, we determined the intrinsic stability time periods (in vitro half-lives) of the encoded ribozymes to be ∼15 days for L1917 and ∼5 days for L1951, possibly due to differences in their sizes (551 and 1,559 bases, respectively), relative degrees of compactness of the respective RNA structures, and amounts of single-stranded RNA. In vivo half-lives for both introns were also determined to be ∼11 min by the use of RNase protection assays and an Escherichia coli model. Intron RNAs were quantified in synchronous cultures of C. burnetii and found to closely parallel those of 16S rRNA; i.e., ribozyme levels significantly increased between days 0 and 3 and then remained stable until 8 days postinfection. Both 16S rRNA and ribozyme levels fell during the stationary and death phases (days 8 to 14). The marked stability of the Coxiella intron RNAs is presumably conferred by their association with ribosomes, a stoichiometric relationship that was determined to be one ribozyme, of either type, per 500 ribosomes. Inaccuracies in splicing (exon 2 skipping) were found to increase during the first 5 days in culture, with a rate of approximately one improperly spliced 23S rRNA per 1.3 million copies. The in vitro efficiency of L1917 intron splicing was significantly enhanced in the presence of a recombinant Coxiella RNA DEAD-box helicase (CBU_0670) relative to that of controls, suggesting that this enzyme may serve as an intron RNA splice facilitator in vivo.

Does cell cycle arrest occur in plant under solar UV-B radiation?

UV-B radiation (280-315 nm) is an integral part of solar radiation and has many harmful effects on plant growth and development. However, the molecular mechanism for the inhibition of plant growth by UV-B remains largely unknown.UV-B radiation induces various responses such as growth inhibition, DNA damage and changes of gene expression. Recently, by using synchronous root tip culture, we found that UV-B modulates the expression of cell cycle regulatory genes through DNA damage. Western blotting analysis revealed that UV-B induced G1-to-S arrest did not correlate with the protein abundance of CDKB1;1 and CYCD3;1 gene regulating proteins, but may with the posttranslational control. We extended the expression analysis of cell cycle related genes based on the published microarray data and the results strengthen our assumption that cell cycle arrest could occur in plant under solar UV-B radiation. Further study is needed to elucidate the relationship between cell cycle regulation and protective pathway induced by low dose of UV-B radiation fundamental molecular mechanism for how plants respond to solar UV-B radiation.

Inhibition of NEDD8-Activating Enzyme Induces Rereplication and Apoptosis in Human Tumor Cells Consistent with Deregulating CDT1 Turnover

Loss of NEDD8-activating enzyme (NAE) function by siRNA knockdown or inhibition by the small molecule NAE inhibitor MLN4924 leads to increased steady-state levels of direct Cullin-RING ligase (CRL) substrates by preventing their ubiquitination and proteasome-dependent degradation. Many of these CRL substrates are involved in cell cycle progression, including a critical DNA replication licensing factor CDT1. Cell cycle analysis of asynchronous and synchronous cultures after NAE inhibition revealed effects on cell cycle distribution and activation of DNA break repair signaling pathways similar to that reported for CDT1 overexpression. The siRNA knockdown of cullins critical for the turnover of CDT1 recapitulated the aberrant rereplication phenotype while CDT1 knockdown was suppressing. Although NAE inhibition leads to deregulation of many CRL substrates, these data demonstrate that CDT1 accumulation mediates the DNA rereplication phenotype resulting from loss of NAE function. DNA rereplication is an unrecoverable cellular insult and the small molecule inhibitor MLN4924, currently in phase I trials, represents an unprecedented opportunity to explore this mechanism of cytotoxicity for the treatment of cancer.

A Fragmenting Hybrid Approach for Targeted Delivery of Multiple Therapeutic Agents to the Malaria Parasite

Artemisinin combination therapies (ACT) represent the current standard of care in the treatment of uncomplicated malaria. The widespread adoption of ACT has been motivated by a desire to minimize the emergence of drug resistance and to address the problem of recrudescence associated with artemisinin monotherapy.1–4 We set out to explore a single-molecule ‘fragmenting hybrid’ strategy in which an artemisinin-like peroxide is employed to deliver a partner drug, only upon activation by ferrous iron in the parasite. In principle, iron(II)-dependent drug delivery from a fragmenting hybrid could alleviate unwanted off-target bioactivities of the partner drug, which would be inactive in its hybrid form.

Acclimation to low or limiting CO2 in non-synchronous Chlamydomonas causes a transient synchronization of the cell division cycle

Acclimation of Chlamydomonas reinhardtii (hereafter, Chlamydomonas) to low or limiting CO(2) or inorganic carbon (C(i)) has been studied fairly extensively with regard to the mechanisms underlying the inducible C(i) acquisition systems and the signal transduction pathway involved in recognizing and responding to decreased C(i) availability. Investigation of low C(i )acclimation responses typically is performed with non-synchronous cultures grown in continuous light to avoid any effects of the cell division cycle (CDC) confounding interpretation of acclimation responses. However, little is known about whether acclimation to low C(i) might affect the distribution of cells among the various stages of the CDC. To investigate the effects of a limiting-C(i) challenge on the CDC of Chlamydomonas, flow cytometry was used to monitor the distribution of cells among the CDC stages in both synchronous and non-synchronous cultures during acclimation to low or limiting C(i). When faced with C(i) limitation, non-synchronous cultures of Chlamydomonas undergo transient synchronization as those cells past the Commitment point of the CDC undergo division, while the remainder of the cells pause their growth in early G-phase, with the result that the cells all accumulate in early G-phase, appearing transiently synchronized until acclimated sufficiently to the decreased C(i) for growth to resume. This perturbation of the CDC by a limiting-C(i) challenge has important implications for the interpretation of gene expression and other responses apparently induced by low or limiting C(i).

RNA-Seq analysis of splicing in Plasmodium falciparum uncovers new splice junctions, alternative splicing and splicing of antisense transcripts

Over 50% of genes in Plasmodium falciparum, the deadliest human malaria parasite, contain predicted introns, yet experimental characterization of splicing in this organism remains incomplete. We present here a transcriptome-wide characterization of intraerythrocytic splicing events, as captured by RNA-Seq data from four timepoints of a single highly synchronous culture. Gene model-independent analysis of these data in conjunction with publically available RNA-Seq data with HMMSplicer, an in-house developed splice site detection algorithm, revealed a total of 977 new 5′ GU-AG 3′ and 5 new 5′ GC-AG 3′ junctions absent from gene models and ESTs (11% increase to the current annotation). In addition, 310 alternative splicing events were detected in 254 (4.5%) genes, most of which truncate open reading frames. Splicing events antisense to gene models were also detected, revealing complex transcriptional arrangements within the parasite’s transcriptome. Interestingly, antisense introns overlap sense introns more than would be expected by chance, perhaps indicating a functional relationship between overlapping transcripts or an inherent organizational property of the transcriptome. Independent experimental validation confirmed over 30 new antisense and alternative junctions. Thus, this largest assemblage of new and alternative splicing events to date in Plasmodium falciparum provides a more precise, dynamic view of the parasite’s transcriptome.

Application of molecular genetic and microbiological techniques in ecology and biotechnology of cyanobacteria

The review discusses the advances and problems in biotechnology and ecology of cyanobacteria and considers the possibilities of molecular genetic and microbiological techniques in this field. Due to the ease of cultivation, high growth rate, availability of synchronous cultures, and existence of numerous molecular genetic and microbiological techniques for various cyanobacterial strains, cyanobacteria—prokaryotic organisms that are ancient relatives of the chloroplasts—are model organisms in the studies of photosynthesis, dinitrogen fixation, cell division, hydrogen production, and in a number of other areas of basic and applied science. These techniques make possible deeper understanding of the role of cyanobacteria in various ecosystems and utilization of their potential in numerous applied projects, including production of molecular hydrogen, phycobiliproteins, and cyanophycin; formation of nanoparticles; removal of heavy metals from the environment; substrate biodegradation; manufacture of products for medicine and food industry; and solution of the problem of cyanobacterial toxins in freshwater and marine environments.

Regulation of the Chlamydomonas cell cycle by a stable, chromatin-associated retinoblastoma tumor suppressor complex.

We examined the cell cycle dynamics of the retinoblastoma (RB) protein complex in the unicellular alga Chlamydomonas reinhardtii that has single homologs for each subunit-RB, E2F, and DP. We found that Chlamydomonas RB (encoded by MAT3) is a cell cycle-regulated phosphoprotein, that E2F1-DP1 can bind to a consensus E2F site, and that all three proteins interact in vivo to form a complex that can be quantitatively immunopurified. Yeast two-hybrid assays revealed the formation of a ternary complex between MAT3, DP1, and E2F1 that requires a C-terminal motif in E2F1 analogous to the RB binding domain of plant and animal E2Fs. We examined the abundance of MAT3/RB and E2F1-DP1 in highly synchronous cultures and found that they are synthesized and remain stably associated throughout the cell cycle with no detectable fraction of free E2F1-DP1. Consistent with their stable association, MAT3/RB and DP1 are constitutively nuclear, and MAT3/RB does not require DP1-E2F1 for nuclear localization. In the nucleus, MAT3/RB remains bound to chromatin throughout the cell cycle, and its chromatin binding is mediated through E2F1-DP1. Together, our data show that E2F-DP complexes can regulate the cell cycle without dissociation of their RB-related subunit and that other changes may be sufficient to convert RB-E2F-DP from a cell cycle repressor to an activator.

MitoGenesisDB: an expression data mining tool to explore spatio-temporal dynamics of mitochondrial biogenesis

Mitochondria constitute complex and flexible cellular entities, which play crucial roles in normal and pathological cell conditions. The database MitoGenesisDB focuses on the dynamic of mitochondrial protein formation through global mRNA analyses. Three main parameters confer a global view of mitochondrial biogenesis: (i) time-course of mRNA production in highly synchronized yeast cell cultures, (ii) microarray analyses of mRNA localization that define translation sites and (iii) mRNA transcription rate and stability which characterize genes that are more dependent on post-transcriptional regulation processes. MitoGenesisDB integrates and establishes cross-comparisons between these data. Several model organisms can be analyzed via orthologous relationships between interspecies genes. More generally this database supports the ‘post-transcriptional operon’ model, which postulates that eukaryotes co-regulate related mRNAs based on their functional organization in ribonucleoprotein complexes. MitoGenesisDB allows identifying such groups of post-trancriptionally regulated genes and is thus a useful tool to analyze the complex relationships between transcriptional and post-transcriptional regulation processes. The case of respiratory chain assembly factors illustrates this point. The MitoGenesisDB interface is available at http://www.dsimb.inserm.fr/dsimb_tools/mitgene/.

New synchronization method for Plasmodium falciparum

BackgroundPlasmodium falciparum is usually asynchronous during in vitro culture. Although various synchronization methods are available, they are not able to narrow the range of ages of parasites. A newly developed method is described that allows synchronization of parasites to produce cultures with an age range as low as 30 minutes.MethodsTrophozoites and schizonts are enriched using Plasmion. The enriched late stage parasites are immobilized as a monolayer onto plastic Petri dishes using concanavalin A. Uninfected erythrocytes are placed onto the monolayer for a limited time period, during which time schizonts on the monolayer rupture and the released merozoites invade the fresh erythrocytes. The overlay is then taken off into a culture flask, resulting in a highly synchronized population of parasites.ResultsPlasmion treatment results in a 10- to 13-fold enrichment of late stage parasites. The monolayer method results in highly synchronized cultures of parasites where invasion has occurred within a very limited time window, which can be as low as 30 minutes. The method is simple, requiring no specialized equipment and relatively cheap reagents.ConclusionsThe new method for parasite synchronization results in highly synchronized populations of parasites, which will be useful for studies of the parasite asexual cell cycle.