Cells In Different Growth Phases Research Articles

Two-signal electrochemical detection system for evaluation viability of Staphylococcus aureus

Establishment of new method for evaluation microbial viability has become an urgent need in the fields of biology, medicine, environment and so on. In this paper, two electrochemical signals of Staphylococcus aureus (S. aureus) at + 0.693 and + 0.973 V vs. Ag/AgCl (sat. KCl) were detected on the multi-walled carbon nanotube modified glassy carbon electrode (MWCNTs/GCE), and attributed to the oxidation of guanine and adenine secreted by S. aureus. For the first time, bacterial viability was evaluated from the perspective of proliferation and purine metabolism. Compared with the plate counting method, the electrochemical signals of guanine and adenine showed an earlier change of viability when S. aureus entered stationary from logarithmic phase and apoptotic from stationary phase during the growth process of S. aureus. There were good linear relationships between electrochemical signals of guanine and adenine and the number of S. aureus cells in different growth phases, but there were different detection thresholds. At the logarithmic phase, S. aureus had stronger viability and ability to metabolize purines, which resulted in a lower detection threshold. Both electrochemical signals of bacteria could well reflect the inhibition of levofloxacin on S. aureus viability, and the guanine signal peak was closer to the result of plate counting method.

Biofuel and bioremediation prospects of estuarine diatoms

Escalating fuel prices, rising environmental concerns and dwindling stocks of fossil fuel have necessitated the exploration of viable alternative feedstocks. Biofuel from microalgae has been showing prospects of a viable alternative to conventional fossil fuels. The present study evaluates the bioremediation and biofuel potential of indigenously collected mixed consortia of estuarine diatoms, grown using aquaculture wastewater as the nutrient source. Diatoms, when subjected to different treatment conditions, exhibited better nutrients removal efficiencies (N: 89.1 ± 0.85%, P: 90.8 ± 0.12%) with significant variations in lipid content (14%-32%). Diatoms depicted real-time accumulation of neutral lipids during different phases of cell growth, observed through flow cytometry. Fatty acid profiling using GC-MS showed properties comparable to that of the plant-based oils. These results show the feasibility of growing diatoms in aquaculture wastewater and in vivo quantification of lipids using flow cytometry.

Scenedesmus obliquus metabolomics: effect of photoperiods and cell growth phases.

Environmental factors directly affect the growth and composition of microalgal biomass. Therefore, the present work analyzed the metabolomics (amino acids, organic acids, and fatty acids) of the microalga Scenedesmus obliquus cultivated in 24:0 and 12:12 (light:dark) photoperiods and different phases of cell growth. Furthermore, the metabolites were related to protein, lipid, and chlorophyll contents at the end of cultivation. The highest biomass concentration (4020mgL- 1) and protein (47.3%) were obtained in culture under constant illumination. The cultivation 12:12 (light:dark) photoperiod triggered higher production of lipids (23.0%) and chlorophylls (26.4mgg- 1) by S. obliquus. Microalgal metabolites were greatly affected by photoperiod and by phase of cell growth. Thus, metabolite production could be related to both the environmental conditions under which cultivation occurred and to the different concentrations of products (proteins, lipids, and chlorophylls) present in the S. obliquus biomass.

Using Raman spectroscopy and chemometrics to identify the growth phase of Lactobacillus casei Zhang during batch culture at the single-cell level

BackgroundAs microbial cultures are comprised of heterogeneous cells that differ according to their size and intracellular concentrations of DNA, proteins, and other constituents, the detailed identification and discrimination of the growth phases of bacterial populations in batch culture is challenging. Cell analysis is indispensable for quality control and cell enrichment.MethodsIn this paper, we report the results of our investigation on the use of single-cell Raman spectrometry (SCRS) for real-time analysis and prediction of cells in different growth phases during batch culture of Lactobacillus (L.) casei Zhang. A targeted analysis of defined cell growth phases at the level of the single cell, including lag phase, log phase, and stationary phase, was facilitated by SCRS.Results Spectral shifts were identified in different states of cell growth that reflect biochemical changes specific to each cell growth phase. Raman peaks associated with DNA and RNA displayed a decrease in intensity over time, whereas protein-specific and lipid-specific Raman vibrations increased at different rates. Furthermore, a supervised classification model (Random Forest) was used to specify the lag phase, log phase, and stationary phase of cells based on SCRS, and a mean sensitivity of 90.7% and mean specificity of 90.8% were achieved. In addition, the correct cell type was predicted at an accuracy of approximately 91.2%.Conclusions To conclude, Raman spectroscopy allows label-free, continuous monitoring of cell growth, which may facilitate more accurate estimates of the growth states of lactic acid bacterial populations during fermented batch culture in industry.

Further investigation of relationships between membrane fluidity and ethanol tolerance in Saccharomyces cerevisiae.

Membrane lipid unsaturation index and membrane fluidity have been related to yeast ethanol stress tolerance in published studies, however findings have been inconsistent. In this study, viability reduction on exposure to 18% (v/v) ethanol was compared to membrane fluidity determined by laurdan generalized polarization. Furthermore, in the determination of viability reduction, we examined the effectiveness of two methods, namely total plate count and methylene violet staining. We found a strong negative correlation between ethanol tolerance and membrane fluidity, indicated by negative Pearson correlation coefficients of - 0.79, - 0.65 and - 0.69 for Saccharomyces cerevisiae strains A12, PDM and K7, respectively. We found that lower membrane fluidity leads to higher ethanol tolerance, as indicated by decreased viability reduction and higher laurdan generalized polarization in respiratory phase compared to respiro-fermentative phase cells. Total plate count better differentiated ethanol tolerance of yeast cells in different growth phases, while methylene violet staining was better to differentiate ethanol tolerance of the different yeast strains at a particular culture phase. Hence, both viability assessment methods have their own advantages and limitations, which should be considered when comparing stress tolerance in different situations.

Optimization of Sucrose Concentration and Light Treatment in Cell Suspension Culture Establishment of Barringtonia racemosa L.

Objective: This study was carried out to optimize the concentration of sucrose and light treatment for the establishment of cell suspension cultures with the greatest records of dry cell biomass. Methods: The homogeneous cell suspension cultures of B. racemosa cultured in MS (Murashige & Skoog’s) media with 1.0 mg/l 2,4-D (2,4-dichlorophenoxyacetic acid) and 1.5 mg/l kinetin were optimized for sucrose concentrations (2.0, 3.0 & 4.0% w/v) and light treatment (24-hour light or 24-hour dark). The dry cell biomass and pH values were recorded for 30 days. The data gathered were plotted against time and statistically analysed by analysis of variance (ANOVA). Results: The supplementation of 4.0% sucrose in 24-hour dark treatment recorded the most optimum dry cell biomass record. The different phases of cell growth could be identified from the growth curve constructed based on dry cell biomass records. The first two-day duration was identified as lag phase in all treatments tested followed by growth exponential phase and constant growth in cells until they achieved their maximum dry cell biomass. Afterwards, some of the treatments underwent rapid declining phase while others showed a short duration of stationary phase by depicting a plateau stage in the cell kinetic plot before finally entering the declining phase afterwards. It has been observed that the cells treated in the dark achieved their highest record of dry cell biomass earlier than those treated in the presence of light in the cultures supplemented with 3.0% and 4.0% sucrose. The effects of variations in sucrose concentration and light treatment were generally significant (p 0.05). Novelty/Improvement: A protocol for the establishment of cell suspension cultures of B. racemosa and the importance of sucrose and light treatments towards cell growth had been developed and highlighted. Keywords: Barringtonia racemosa L, Cell Suspension Culture, Light Treatment, Sucrose Concentration

Growth Phase, Oxygen, Temperature, and Starvation Affect the Development of Viable but Non-culturable State of Vibrio cholerae.

Vibrio cholerae can enter into a viable but non-culturable (VBNC) state in order to survive in unfavorable environments. In this study, we studied the roles of five physicochemical and microbiological factors or states, namely, different strains, growth phases, oxygen, temperature, and starvation, on the development of VBNC of V. cholerae in artificial sea water (ASW). Different strains of the organism, the growth phase, and oxygen levels affected the progress of VBNC development. It was found that the VBNC state was induced faster in V. cholerae serogroup O1 classical biotype strain O395 than in O1 El Tor biotype strains C6706 and N16961. When cells in different growth phases were used for VBNC induction, stationary-phase cells lost their culturability more quickly than exponential-phase cells, while induction of a totally non-culturable state took longer to achieve for stationary-phase cells in all three strains, suggesting that heterogeneity of cells should be considered. Aeration strongly accelerated the loss of culturability. During the development of the VBNC state, the culturable cell count under aeration conditions was almost 106-fold lower than under oxygen-limited conditions for all three strains. The other two factors, temperature and nutrients-rich environment, may prevent the induction of VBNC cells. At 22 or 37°C in ASW, most of the cells rapidly died and the culturable cell count reduced from about 108 to 106–105 CFU/mL. The total cell counts showed that cells that lost viability were decomposed, and the viable cell counts were the same as culturable cell counts, indicating that the cells did not reach the VBNC state. VBNC state development was blocked when ASW was supplied with Luria-Bertani broth (LB), but it was not affected in ASW with M9, suggesting that specific nutrients in LB may prevent the development of VBNC state. These results revealed that the five factors evaluated in this study had different roles during the progress of VBNC induction. Changing a single factor could influence and even block the development of the VBNC state. These findings provide new insight to help design further studies to better understand the mechanisms which trigger the development and regulation of the VBNC state.

Graphene oxide and titanium: synergistic effects on the biomineralization ability of osteoblast cultures.

Graphene oxide (GO) has attracted remarkable attention in recent years due to properties such as extremely large surface area, biocompatibility, biostability, and easy chemical functionalization. Osteoblasts underlie the deposition of hydroxyapatite crystals in the bone protein matrix during biomineralization; hydroxyapatite deposition involves extracellular matrix vesicles that are rich in alkaline phosphatase (ALP). Here, we have investigated how GO affects osteoblast viability, ALP activity, and mineralized matrix formation in osteoblast cultures in three different phases of cell growth, in the presence and in the absence of titanium (Ti). Scanning electron microscopy (SEM), Raman spectra, and energy dispersive spectroscopy aided GO characterization. The presence of GO increased the viability of osteoblast cells grown on a plastic surface. However, osteoblast viability on Ti discs was lower in the presence than in the absence of GO. ALP activity emerged at 14 days for the cell culture incubated with GO. The total protein concentration also increased at 21 days on both the Ti discs and plastic surface. Osteoblasts grown on Ti discs had increased mineralized matrix formation in the presence of GO as compared to the cells grown in the absence of GO. SEM images of the cell cultures on plastic surfaces in the presence of GO suggested delayed mineralized matrix formation. In conclusion, applications requiring the presence of Ti, such as prostheses and implants, should benefit from the use of GO, which may increase mineralized nodule formation, stimulate biomineralization, and accelerate bone regeneration.

Phenotypic diversity of diploid and haploid Emiliania huxleyi cells and of cells in different growth phases revealed by comparative metabolomics

In phytoplankton a high species diversity of microalgae co-exists at a given time. But diversity is not only reflected by the species composition. Within these species different life phases as well as different metabolic states can cause additional diversity. One important example is the coccolithophore Emiliania huxleyi. Diploid cells play an important role in marine ecosystems since they can form massively abundant algal blooms but in addition the less abundant haploid life phase of E. huxleyi occurs in lower quantities. Both life phases may fulfill different functions in the plankton. We hypothesize that in addition to the functional diversity caused by this life phase transition the growth stage of cells can also influence the metabolic composition and thus the ecological impact of E. huxleyi. Here we introduce a metabolomic survey in dependence of life phases as well as different growth phases to reveal such changes. The comparative metabolomic approach is based on the extraction of intracellular metabolites from intact microalgae, derivatization and analysis by gas chromatography coupled to mass spectrometry (GC-MS). Automated data processing and statistical analysis using canonical analysis of principal coordinates (CAP) revealed unique metabolic profiles for each life phase. Concerning the correlations of metabolites to growth phases, complex patterns were observed. As for example the saccharide mannitol showed its highest concentration in the exponential phase, whereas fatty acids were correlated to stationary and sterols to declining phase. These results are indicative for specific ecological roles of these stages of E. huxleyi and are discussed in the context of previous physiological and ecological studies.

Purine metabolite and energy charge analysis of Trypanosoma brucei cells in different growth phases using an optimized ion-pair RP-HPLC/UV for the quantification of adenine and guanine pools

Human African Trypanosomiasis (HAT) is caused by the protozoan parasite Trypanosoma brucei. Although trypanosomes are well-studied model organisms, only little is known about their adenine and guanine nucleotide pools. Besides being building blocks of RNA and DNA, these nucleotides are also important modulators of diverse biochemical cellular processes. Adenine nucleotides also play an important role in the regulation of metabolic energy. The energetic state of cells is evaluated by the energy charge which gives information about how much energy is available in form of high energy phosphate bonds of adenine nucleotides. A sensitive and reproducible ion-pair RP-HPLC/UV method was developed and optimized, allowing the quantification of guanine and adenine nucleosides/nucleotides in T. brucei. With this method, the purine levels and their respective ratios were investigated in trypanosomes during logarithmic, stationary and senescent growth phases. Results of this study showed that all adenine and guanine purines under investigation were in the low mM range. The energy charge was found to decrease from logarithmic to static and to senescent phase whereas AMP/ATP, ADP/ATP and GDP/GTP ratios increased in the same order. In addition, the AMP/ATP ratio varied as the square of the ADP/ATP ratio, indicating AMP to be the key energy sensor molecule in trypanosomes.

Ion mobility–mass spectrometry of a rotary ATPase reveals ATP-induced reduction in conformational flexibility

Rotary ATPases play fundamental roles in energy conversion as their catalytic rotation is associated with interdomain fluctuations and heterogeneity of conformational states. Using ion mobility mass spectrometry we compared the conformational dynamics of the intact ATPase from Thermus thermophilus with those of its membrane and soluble subcomplexes. Our results define regions with enhanced flexibility assigned to distinct subunits within the overall assembly. To provide a structural context for our experimental data we performed molecular dynamics simulations and observed conformational changes of the peripheral stalks that reflect their intrinsic flexibility. By isolating complexes at different phases of cell growth and manipulating nucleotides, metal ions and pH during isolation, we reveal differences that can be related to conformational changes in the Vo complex triggered by ATP binding. Together these results implicate nucleotides in modulating flexibility of the stator components and uncover mechanistic detail that underlies operation and regulation in the context of the holoenzyme.

SUN family proteins Sun4p, Uth1p and Sim1p are secreted from Saccharomyces cerevisiae and produced dependently on oxygen level.

The SUN family is comprised of proteins that are conserved among various yeasts and fungi, but that are absent in mammals and plants. Although the function(s) of these proteins are mostly unknown, they have been linked to various, often unrelated cellular processes such as those connected to mitochondrial and cell wall functions. Here we show that three of the four Saccharomyces cerevisiae SUN family proteins, Uth1p, Sim1p and Sun4p, are efficiently secreted out of the cells in different growth phases and their production is affected by the level of oxygen. The Uth1p, Sim1p, Sun4p and Nca3p are mostly synthesized during the growth phase of both yeast liquid cultures and colonies. Culture transition to slow-growing or stationary phases is linked with a decreased cellular concentration of Sim1p and Sun4p and with their efficient release from the cells. In contrast, Uth1p is released mainly from growing cells. The synthesis of Uth1p and Sim1p, but not of Sun4p, is repressed by anoxia. All four proteins confer cell sensitivity to zymolyase. In addition, Uth1p affects cell sensitivity to compounds influencing cell wall composition and integrity (such as Calcofluor white and Congo red) differently when growing on fermentative versus respiratory carbon sources. In contrast, Uth1p is essential for cell resistance to boric acids irrespective of carbon source. In summary, our novel findings support the hypothesis that SUN family proteins are involved in the remodeling of the yeast cell wall during the various phases of yeast culture development and under various environmental conditions. The finding that Uth1p is involved in cell sensitivity to boric acid, i.e. to a compound that is commonly used as an important antifungal in mycoses, opens up new possibilities of investigating the mechanisms of boric acid’s action.

An ultra scale‐down approach to study the interaction of fermentation, homogenization, and centrifugation for antibody fragment recovery from rec E. coli

Escherichia coli is frequently used as a microbial host to express recombinant proteins but it lacks the ability to secrete proteins into medium. One option for protein release is to use high-pressure homogenization followed by a centrifugation step to remove cell debris. While this does not give selective release of proteins in the periplasmic space, it does provide a robust process. An ultra scale-down (USD) approach based on focused acoustics is described to study rec E. coli cell disruption by high-pressure homogenization for recovery of an antibody fragment (Fab') and the impact of fermentation harvest time. This approach is followed by microwell-based USD centrifugation to study the removal of the resultant cell debris. Successful verification of this USD approach is achieved using pilot scale high-pressure homogenization and pilot scale, continuous flow, disc stack centrifugation comparing performance parameters such as the fraction of Fab' release, cell debris size distribution and the carryover of cell debris fine particles in the supernatant. The integration of fermentation and primary recovery stages is examined using USD monitoring of different phases of cell growth. Increasing susceptibility of the cells to disruption is observed with time following induction. For a given recovery process this results in a higher fraction of product release and a greater proportion of fine cell debris particles that are difficult to remove by centrifugation. Such observations are confirmed at pilot scale.

Dynamic Evaluation of Cell Surface N-Glycan Expression via an Electrogenerated Chemiluminescence Biosensor Based on Concanavalin A-Integrating Gold-Nanoparticle-Modified Ru(bpy)32+-Doped Silica Nanoprobe

A sandwich electrogenerated chemiluminescence (ECL) biosensor was fabricated based on concanavalin A (Con A)-integrating gold-nanoparticle-modified Ru(bpy)3(2+)-doped silica nanoprobe (Au-RuSiO2 NPs) for in situ and dynamically evaluating cell surface N-glycan expression. Owing to the specific recognition of Con A with mannose and the core trimannoside fragment of N-glycan and the effective ECL amplification of Au-RuSiO2 NPs, the as-proposed biosensor exhibited excellent analytical performance toward the cytosensing of K562 cells with a wide detection linear range from 1.0 × 10(3) to 1.0 × 10(7) cells mL(-1) and a detection limit of 600 cells mL(-1). More importantly, the strategy was successfully applied to evaluate cell surface N-glycan expression under different external stimuli of inhibitors and enzyme. This biosensor is endowed with feasibility and reliability of generating sensitive insight into the majority of N-glycan expression on the cell surface. Furthermore, the biosensor was employed to dynamically profile cell surface N-glycan expression at different phases of cell growth in vitro. This biosensor is promising in studying and elucidating the N-glycan function in biological and physiological processes.

Inhibition of poly(ADP-ribose) Polymerase Interferes with Trypanosoma cruzi Infection and Proliferation of the Parasite

Poly(ADP-ribosylation) is a post-translational covalent modification of proteins catalyzed by a family of enzymes termed poly(ADP-ribose) polymerases (PARPs). In the human genome, 17 different genes have been identified that encode members of the PARP superfamily. Poly (ADP-ribose) metabolism plays a role in a wide range of biological processes. In Trypanosoma cruzi, PARP enzyme appears to play a role in DNA repair mechanisms and may also be involved in controlling the different phases of cell growth. Here we describe the identification of potent inhibitors for T. cruzi PARP with a fluorescence-based activity assay. The inhibitors were also tested on T. cruzi epimastigotes, showing that they reduced ADP-ribose polymer formation in vivo. Notably, the identified inhibitors are able to reduce the growth rate of T. cruzi epimastigotes. The best inhibitor, Olaparib, is effective at nanomolar concentrations, making it an efficient chemical tool for chacterization of ADP-ribose metabolism in T. cruzi. PARP inhibition also decreases drastically the amount of amastigotes but interestingly has no effect on the amount of trypomastigotes in the cell culture. Knocking down human PARP-1 decreases both the amount of amastigotes and trypomastigotes in cell culture, indicating that the effect would be mainly due to inhibition of human PARP-1. The result suggests that the inhibition of PARP could be a potential way to interfere with T. cruzi infection.

The taste of diatoms: the role of diatom growth phase characteristics and associated bacteria for benthic copepod grazing

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 67:47-58 (2012) - DOI: https://doi.org/10.3354/ame01587 The taste of diatoms: the role of diatom growth phase characteristics and associated bacteria for benthic copepod grazing Marleen De Troch1,*, Inge Vergaerde1,3, Clio Cnudde1, Pieter Vanormelingen2, Wim Vyverman2, Magda Vincx1 1Marine Biology and 2Protistology and Aquatic Ecology, Biology Department, Ghent University, Campus Sterre, Krijgslaan 281 − S8, Ghent 9000, Belgium 3University College West-Flanders, Rijselstraat 5, Brugge 8200, Belgium *Email: marleen.detroch@ugent.be ABSTRACT: The interactions between primary producers and their consumers are of particular interest for the overall functioning of marine ecosystems. The biochemical composition of the organisms involved affects the efficiency of energy transfer in marine food webs. In addition to top-down control by grazers, bottom-up control of these interactions by primary producers and associated bacteria has recently received more attention. Planktonic copepods selectively feed on older diatom cells, a behaviour regulated by changes in exometabolites around diatoms. To test whether this also applies to benthic copepods, Seminavis robusta cells in lag, exponential and stationary growth phases were biochemically screened, and the diversity of associated bacteria was assessed. The diatoms were subsequently 13C prelabelled and offered to the harpacticoid copepod Microarthridion littorale in a grazing experiment. Harpacticoid copepods incorporated more carbon from younger diatom cells in the lag growth phase, which might be based on (1) biochemical differences of diatom cells in different growth phases and (2) the bacteria associated with the diatoms. The younger diatom cells were characterised by a higher C:N ratio and more extracellular polymer secretions but a lower fatty acid content. The bacterial community on these cells differed from those on cells in the later growth phases. Our results thus suggest that the feeding strategies of benthic harpacticoid copepods differ from those of calanoid copepods. This outcome can be explained by the tight contact between benthic copepods and the typical carbon and bacteria-rich biofilm on sediments. KEY WORDS: Diatoms · Grazing · Harpacticoid copepods Full text in pdf format PreviousNextCite this article as: De Troch M, Vergaerde I, Cnudde C, Vanormelingen P, Vyverman W, Vincx M (2012) The taste of diatoms: the role of diatom growth phase characteristics and associated bacteria for benthic copepod grazing. Aquat Microb Ecol 67:47-58. https://doi.org/10.3354/ame01587 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 67, No. 1. Online publication date: September 11, 2012 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2012 Inter-Research.

Adhesion of yeast cells to different porous supports, stability of cell-carrier systems and formation of volatile by-products

The aim of our research was to study how the conditions of immobilization influence cell attachment to two different ceramic surfaces: hydroxylapatite and chamotte tablets. Three fermentative yeast strains, namely brewery TT, B4 (ale, lager) and distillery Bc15a strains belonging to Saccharomyces spp., and one strain of Debaryomyces occidentalis Y500/5 of weak fermentative nature, but with high amylolytic activity due to extracellular α-amylase and glucoamylase, were used in this study. Different media, including cell starvation, were applied for immobilization of yeast strains as well as different phases of cell growth. Immobilization of selected yeasts on a hydroxylapatite carrier was rather weak. However, when incubation of starved yeast cells was conducted in the minimal medium supplemented by calcium carbonate, the scale of immobilization after 24 h was higher, especially for the D. occidentalis strain. Adhesion to hydroxylapatite carriers in wort broth was of reversible character and better results of adhesion were observed in the case of another ceramic carrier-chamotte. The number of immobilized cells was about 106–107 per tablet and cell adhesion was stable during the whole fermentation process. The comparison of the volatile products that were formed during fermentation did not show any significant qualitative and quantitative differences between the free and the immobilized cells. This is the first time when a cheap, porous chamotte surface has been applied to yeast adhesion and fermentation processes.

Poly(ADP-ribose) polymerase plays a differential role in DNA damage-response and cell death pathways in Trypanosoma cruzi

Poly(ADP-ribosyl)ation is a post-translational modification of proteins. Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are the enzymes responsible for poly(ADP-ribose) (PAR) polymer metabolism and are present in most higher eukaryotes. The best understood role of PARP is the maintenance of genomic integrity either via promotion of DNA repair at low levels of genotoxic stress or via promotion of cell death at higher levels of damage. The unicellular eukaryote Trypanosoma cruzi, as opposed to humans and other organisms, has only one PARP (TcPARP) and one PARG (TcPARG). In the present study we show that under different DNA-damaging agents (H2O2 or UV-C radiation) TcPARP is activated and translocated from the cytosol to the nucleus, while TcPARG always shows a nuclear localisation. Parasites in the presence of PARP or PARG inhibitors, as well as parasites over-expressing either TcPARP or TcPARG, suggested that PAR metabolism could be involved in different phases of cell growth, even in the absence of DNA damage. We also believe that we provide the first reported evidence that different proteins could be poly(ADP-ribosyl)ated in response to different stimuli, leading to different cell death pathways.

Rapid FlAsH labelling in the budding yeast Saccharomyces cerevisiae

Live cell imaging of protein distributions is an essential tool in modern cell biology. It relies on the functional labelling of a host protein with a fluorophore, which may either be a genetically fused fluorescent protein or an organic dye binding to the host protein. The biarsenical-tetracysteine system or 'FlAsH-labelling', is based on the high affinity interaction between a biarsenical probe and a small protein tag. This approach has been successfully used for live cell imaging in the budding yeast Saccharomyces cerevisiae. However, the established labelling protocols require a lengthy overnight incubation of the cells with the dye under tightly controlled growth conditions, which severely limits the use of this approach. In this study, we characterize an efficient method for introducing FlAsH-EDT(2) into live budding yeast cells using standard electroporation. The labelling time is reduced from more than 12 h to less than 1 h without compromising the labelling efficiency or cell viability. This approach may be used for cells in different growth phases or grown under different conditions. It may be further extended to other small high affinity probes, thus opening up new possibilities for labelling in budding yeast.

The steady-states of a multi-compartment, age–size distribution model of cell-growth

A model of cell-growth, describing the evolution of the age–size distribution of cells in different phases of cell-growth, is studied. The model is based on that used in several papers by Basse et al. and is composed of a system of partial differential equations, each describing the changes in the age–size distribution of cells in a specific phase of cell-growth. Here, the ‘age’ of a cell is considered to be the time spent in its current phase of cell-growth, while ‘size’ is considered to be the DNA content of the cell. The existence of steady age–size distributions (SASDs), where the age–size distributions retain the same shape but are scaled up or down as time increases, is investigated and it is shown that SASDs exist. A speculative discussion of the stability of these SASDs is also included, but their stability is not conclusively proved.