Nutrient-limited Cells Research Articles

Post-Transcriptional Regulation of Rab7a in Lysosomal Positioning and Drug Resistance in Nutrient-Limited Cancer Cells.

Limited nutrient availability in the tumor microenvironment can cause the rewiring of signaling and metabolic networks to confer cancer cells with survival advantages. We show here that the limitation of glucose, glutamine and serum from the culture medium resulted in the survival of a population of cancer cells with high viability and capacity to form tumors in vivo. These cells also displayed a remarkable increase in the abundance and size of lysosomes. Moreover, lysosomes were located mainly in the perinuclear region in nutrient-limited cells; this translocation was mediated by a rapid post-transcriptional increase in the key endolysosomal trafficking protein Rab7a. The acidic lysosomes in nutrient-limited cells could trap weakly basic drugs such as doxorubicin, mediating resistance of the cells to the drug, which could be partially reversed with the lysosomal inhibitor bafilomycin A1. An in vivo chorioallantoic membrane (CAM) assay indicated a remarkable decrease in microtumor volume when nutrient-limited cells were treated with 5-Fluorouracil (5-FU) and bafilomycin A1 compared to cells treated with either agent alone. Overall, our data indicate the activation of complementary pathways with nutrient limitation that can enable cancer cells to survive, proliferate and acquire drug resistance.

Metabolic impacts of climate change on marine ecosystems: Implications for fish communities and fisheries

AbstractAimClimate change will reshape marine ecosystems over the 21st century through diverse and complex mechanisms that are difficult to assess quantitatively. Here, we characterize expectations for how marine community biomass will respond to the energetic consequences of changes in primary production and temperature‐dependent metabolic rates, under a range of fishing/conservation scenarios.LocationGlobal ocean.Time period1950–2100.Major taxa studiedCommercially harvested marine ectotherms (‘fish’).MethodsWe use a size‐structured macroecological model of the marine ecosystem, coupled with a catch model that allows for calibration with global historical data and simulation of fishing. We examine the four energetic mechanisms that, within the model framework, determine the community response to climate change: net primary production, phytoplankton cell size, and the temperature dependencies of growth and natural mortality.ResultsClimate change decreases the modelled global fish community biomass by as much as 30% by 2100. This results from a diminished energy supply to upper trophic levels as photosynthesis becomes more nutrient limited and phytoplankton cells shrink, and from a temperature‐driven increase of natural mortality that, together, overwhelm the effect of accelerated somatic growth rates. Ocean circulation changes drive regional variations of primary production, producing patterns of winners and losers that largely compensate each other when averaged globally, whereas decreasing phytoplankton size drives weaker but more uniformly negative changes. The climate impacts are similar across the range of conservation scenarios but are slightly amplified in the strong conservation scenarios owing to the greater role of natural mortality.Main conclusionsThe spatial pattern of climate impacts is mostly determined by changes in primary production. The overall decline of community biomass is attributed to a temperature‐driven increase of natural mortality, alongside an overall decrease in phytoplankton size, despite faster somatic growth. Our results highlight the importance of the competition between accelerated growth and mortality in a warming ocean.

Novel RpoS-Dependent Mechanisms Strengthen the Envelope Permeability Barrier during Stationary Phase.

Gram-negative bacteria are intrinsically resistant to detergents and many antibiotics due to synergistic activities of a strong outer membrane (OM) permeability barrier and efflux pumps that capture and expel toxic molecules eluding the barrier. When the bacteria are depleted of an essential nutrient, a program of gene expression providing cross-protection against many stresses is induced. Whether this program alters the OM to further strengthen the barrier is unknown. Here, we identify novel pathways dependent on the master regulator of stationary phase that further strengthen the OM permeability barrier during nutrient limitation, circumventing the need for efflux pumps. Decreased permeability of nutrient-limited cells to toxic compounds has important implications for designing new antibiotics capable of targeting Gram-negative bacteria that may be in a growth-limited state.

Assessing spatial and temporal variability of phytoplankton communities' composition in the Iroise Sea ecosystem (Brittany, France): A 3D modeling approach. Part 1: Biophysical control over plankton functional types succession and distribution

Understanding the dynamic interplay between physical, biogeochemical and biological processes represents a key challenge in oceanography, particularly in shelf seas where complex hydrodynamics are likely to drive nutrient distribution and niche partitioning of phytoplankton communities. The Iroise Sea includes a tidal front called the ‘Ushant Front’ that undergoes a pronounced seasonal cycle, with a marked signal during the summer. These characteristics as well as relatively good observational sampling make it a region of choice to study processes impacting phytoplankton dynamics. This innovative modeling study employs a phytoplankton-diversity model, coupled to a regional circulation model to explore mechanisms that alter biogeography of phytoplankton in this highly dynamic environment. Phytoplankton assemblages are mainly influenced by the depth of the mixed layer on a seasonal time scale. Indeed, solar incident irradiance is a limiting resource for phototrophic growth and small phytoplankton cells are advantaged over larger cells. This phenomenon is particularly relevant when vertical mixing is intense, such as during winter and early spring. Relaxation of wind-induced mixing in April causes an improvement of irradiance experienced by cells across the whole study area. This leads, in late spring, to a competitive advantage of larger functional groups such as diatoms as long as the nutrient supply is sufficient. This dominance of large, fast-growing autotrophic cells is also maintained during summer in the productive tidally-mixed shelf waters. In the oligotrophic surface layer of the western part of the Iroise Sea, small cells coexist in a greater proportion with large, nutrient limited cells. The productive Ushant tidal front's region (1800mgC·m−2·d−1 between August and September) is also characterized by a high degree of coexistence between three functional groups (diatoms, micro/nano-flagellates and small eukaryotes/cyanobacteria). Consistent with previous studies, the biogeography of phytoplankton functional types at the Ushant front during summer displays an intermediate community composition between contrasted sub-regions on either side of the front. Strong mixing conditions within the frontal sub-region result in a short residence time of water masses, not allowing speciation or long term adaptation to occur.

Methanothermobacter thermautotrophicus modulates its membrane lipids in response to hydrogen and nutrient availability.

Methanothermobacter thermautotrophicus strain ΔH is a model hydrogenotrophic methanogen, for which extensive biochemical information, including the complete genome sequence, is available. Nevertheless, at the cell membrane lipid level, little is known about the responses of this archaeon to environmental stimuli. In this study, the lipid composition of M. thermautotrophicus was characterized to verify how this archaeon modulates its cell membrane components during growth phases and in response to hydrogen depletion and nutrient limitation (potassium and phosphate). As opposed to the higher abundance of phospholipids in the stationary phase of control experiments, cell membranes under nutrient, and energy stress were dominated by glycolipids that likely provided a more effective barrier against ion leakage. We also identified particular lipid regulatory mechanisms in M. thermautotrophicus, which included the accumulation of polyprenols under hydrogen-limited conditions and an increased content of sodiated adducts of lipids in nutrient-limited cells. These findings suggest that M. thermautotrophicus intensely modulates its cell membrane lipid composition to cope with energy and nutrient availability in dynamic environments.

Hybrid cellular automaton modeling of nutrient modulated cell growth in tissue engineering constructs

Mathematic models help interpret experimental results and accelerate tissue engineering developments. We develop in this paper a hybrid cellular automata model that combines the differential nutrient transport equation to investigate the nutrient limited cell construct development for cartilage tissue engineering. Individual cell behaviors of migration, contact inhibition and cell collision, coupled with the cell proliferation regulated by oxygen concentration were carefully studied. Simplified two-dimensional simulations were performed. Using this model, we investigated the influence of cell migration speed on the overall cell growth within in vitro cell scaffolds. It was found that intense cell motility can enhance initial cell growth rates. However, since cell growth is also significantly modulated by the nutrient contents, intense cell motility with conventional uniform cell seeding method may lead to declined cell growth in the final time because concentrated cell population has been growing around the scaffold periphery to block the nutrient transport from outside culture media. Therefore, homogeneous cell seeding may not be a good way of gaining large and uniform cell densities for the final results. We then compared cell growth in scaffolds with various seeding modes, and proposed a seeding mode with cells initially residing in the middle area of the scaffold that may efficiently reduce the nutrient blockage and result in a better cell amount and uniform cell distribution for tissue engineering construct developments.

The algal toxicity of silver engineered nanoparticles and detoxification by exopolymeric substances

In this study, we report that silver ions (Ag +) from the oxidative dissolution of silver engineered nanoparticles (Ag-ENs) determined the EN toxicity to the marine diatom Thalassiosira weissflogii. Most of the Ag-ENs formed non-toxic aggregates (>0.22 μm) in seawater. When the free Ag + concentration ([Ag +] F) was greatly reduced by diafiltration or thiol complexation, no toxicity was observed, even though the Ag-ENs were better dispersed in the presence of thiols with up to 1.08 × 10 −5 M Ag-ENs found in the <0.22 μm fraction, which are orders of magnitude higher than predicted for the natural aquatic environment. The secretion of polysaccharide-rich algal exopolymeric substances (EPS) significantly increased at increasing [Ag +] F. Both dissolved and particulate polysaccharide concentrations were higher for nutrient-limited cells, coinciding with their higher Ag + tolerance, suggesting that EPS may be involved in Ag + detoxification.

Effect of Limited Nutrition on In Situ Intervertebral Disc Cells Under Simulated-Physiological Loading

Whole ovine caudal intervertebral discs (IVD) were cultured in sufficient and limited nutrition under simulated-physiologic loading for 7 and 21 days. To study the effect of limited nutrition on disc cells embedded in their native tissue in short- and midterm whole organ disc culture. Nutrient-limited induction of disc cell death in vitro has been demonstrated and is believed to be a factor in disc degeneration. Nutrient-limited cell death and its consequences, as it relates to degeneration, have not been investigated in the intact IVD. Ovine IVDs with endplates were cultured for 7 and 21 days under simulated-physiologic loading, either in media with limited (2 g/L) or sufficient (4.5 g/L) glucose concentration. Cell viability, relative gene expression, newly synthesized chondroitin sulfate content, and matrix metalloproteinase (MMP) activity were measured after culture and compared to fresh tissue. In sufficient glucose media, cell viability was maintained through 7 days to 21 days of culture. In limited glucose, it dropped significantly to 62% in the anulus fibrosus and to 56% in the nucleus pulposus after 7 days and remained so until 21 days (63% in the anulus fibrosus and 52% in the nucleus pulposus). No significant differences were found between culture conditions for relative gene expression, newly synthesized chondroitin sulfate and inactive and active forms of MMP13 and MMP7. With this culture system, whole IVD explants could be maintained up to 21 days. Cell viability decreased to 50% to 60% under limited nutrition within days and remained so up to 3 weeks. The surviving cells did not compensate matrix production in this time frame.

Discoveries in Bacterial Nucleotide Metabolism

Discoveries in Bacterial Nucleotide Metabolism

Microbial community development in the traps of aquatic Utricularia species

We examined trap fluid of three aquatic carnivorous species of Utricularia (Lentibulariaceae) to assess the role of microbial community within their traps in plant nutrient acquisition. In the context of increasing trap age, we characterized microbial community composition using phospholipid fatty acid (PLFA) analysis and microscopy. Nutrient content in various fractions of the trap fluid was analyzed and the abundance of free-suspended bacteria estimated. The activities of extracellular phosphatase in the trap fluid were determined using fluorometry and the contribution of the microbial community to phosphatase production assessed by epifluorescence microscopy. The trap microbial community seems to be largely derived from Utricularia associated periphyton. PLFA analysis revealed that trap fluid contained all components of a complex microbial food web with bacteria forming more than 58% of the viable microbial biomass in the trap. Trap age seems to be the key factor in determining the patterns of microbial community development as well as enzyme production. The amount of nutrients increases with increasing trap age, and the total amounts of C, N, and P accumulated within traps during their lifetime are relatively large—of the order of 100 mg L −1 for C and N, and between 0.2 and 0.6 mg L −1 for P. A significant part of the nutrient pool is present in the dissolved form. Trap fluid stoichiometry (molar N:P ratios about 100) as well as the presence of nutrient limited microbial cells (molar N:P ratios 25–61) indicates the importance of phosphorus rather than nitrogen for the nutrition of Utricularia. Our findings support the hypothesis that mutualism, apart from the predator–prey interaction, is an important association in aquatic Utricularia traps and that the trap-associated microbial community may be of benefit to the rootless aquatic Utricularia species facing problems with P acquisition due to the loss of roots in their evolution.

Enhancement of cell growth in tissue‐engineering constructs under direct perfusion: Modeling and simulation

Perfusion bioreactors improve mass transfer in cell-scaffold constructs. We developed a mathematical model to simulate nutrient flow through cellular constructs. Interactions among cell proliferation, nutrient consumption, and culture medium circulation were investigated. The model incorporated modified Contois cell-growth kinetics that includes effects of nutrient saturation and limited cell growth. Nutrient uptake was depicted through the Michaelis-Menton kinetics. To describe the culture medium convection, the fluid flow outside the cell-scaffold construct was described by the Navier-Stokes equations, while the fluid dynamics within the construct was modeled by Brinkman's equation for porous media flow. Effects of the media perfusion were examined by including time-dependant porosity and permeability changes due to cell growth. The overall cell volume was considered to consist of cells and extracellular matrices (ECM) as a whole without treating ECM separately. Numerical simulations show when cells were cultured subjected to direct perfusion, they penetrated to a greater extent into the scaffold and resulted in a more uniform spatial distribution. The cell amount was increased by perfusion and ultimately approached an asymptotic value as the perfusion rates increased in terms of the dimensionless Peclet number that accounts for the ratio of nutrient perfusion to diffusion. In addition to enhancing the nutrient delivery, perfusion simultaneously imposes flow-mediated shear stress to the engineered cells. Shear stresses were found to increase with cell growth as the scaffold void space was occupied by the cell and ECM volumes. The macro average stresses increased from 0.2 mPa to 1 mPa at a perfusion rate of 20 microm/s with the overall cell volume fraction growing from 0.4 to 0.7, which made the overall permeability value decrease from 1.35 x 10(-2)cm(2) to 5.51 x 10(-4)cm(2). Relating the simulation results with perfusion experiments in literature, the average shear stresses were below the critical value that would induce the chondrocyte necrosis.

Effect of Process Variables on Supercritical Fluid Disruption of Ralstonia eutropha Cells for Poly(R-hydroxybutyrate) Recovery

This research focuses on the disruption of the gram-negative bacterium Ralstonia eutropha cells by supercritical CO2 for poly(R-hydroxybutyrate) (PHB) recovery. The variables affecting cell disruption such as drying strategy, type of modifier, and cultivation time, as well as operating pressure, temperature, and repeated release of supercritical CO2 pressure, have been studied. Effect of this disruption technique on PHB molecular mass was also investigated. PHB recovery was examined using a combination of this method and chemical pretreatments. For salt pretreatment, the cells were exposed to 140 mM NaCl and heat (60 degrees C, 1 h). The cells were also exposed to 0.2-0.8% (w/w) NaOH to examine the effect of alkaline pretreatment. Bacterial cells treated in growth phase exhibited less resistance to disruption than nutrient-limited cells in the stationary phase. It was also found that the wet cells could be utilized to recover PHB, but purity of the product was lower than that obtained from freeze-dried cells. Pretreatment with a minimum of 0.4% (w/w) NaOH was necessary to enable complete disruption with two times pressure release. Salt pretreatment was less effective; however, disruption was improved by the application of alkaline shock. The proposed method is economic and comparable with other recovery methods in terms of the percentage of PHB recovery and energy consumption, while it is environmentally more benign.

Redfield revisited: variability of C:N:P in marine microalgae and its biochemical basis

A compilation of data on the elemental composition of marine phytoplankton from published studies was used to determine the range of C:N:P. The N:P ratio of algae and cyanobacteria is very plastic in nutrient-limited cells, ranging from <5 mol N:mol P when phosphate is available greatly in excess of nitrate or ammonium to <100 mol N:mol P when inorganic N is present greatly in excess of P. Under optimal nutrient-replete growth conditions, the cellular N:P ratio is somewhat more constrained, ranging from 5 to 19 mol N:mol P, with most observations below the Redfield ratio of 16. Limited data indicate that the critical N:P that marks the transition between N- and P-limitation of phytoplankton growth lies in the range 20–50 mol N:mol P, considerably in excess of the Redfield ratio. Biochemical composition can be used to constrain the critical N:P. Although the biochemical data do not preclude the critical N:P from being as high as 50, the typical biochemical composition of nutrient-replete algae and cyanobacteria suggests that the critical N:P is more likely to lie in the range between 15 and 30. Despite the observation that the overall average N:P composition of marine particulate matter closely approximates the Redfield ratio of 16, there are significant local variations with a range from 5 to 34. Consistent with the culture studies, lowest values of N:P are associated with nitrate- and phosphate-replete conditions. The highest values of N:P are observed in oligotrophic waters and are within the range of critical N:P observed in cultures, but are not so high as to necessarily invoke P-limitation. The C:N ratio is also plastic. The average C:N ratios of nutrientreplete phytoplankton cultures, oceanic particulate matter and inorganic N and C draw-down are slightly greater than the Redfield ratio of 6.6. Neither the analysis of laboratory C:N:P data nor a more theoretical approach based on the relative abundance of the major biochemical molecules in the phytoplankton can support the contention that the Redfield N:P reflects a physiological or biochemical constraint on the elemental composition of primary production.

Approaches for determining phytoplankton nutrient limitation

Aquatic primary productivity is frequently limited by the availability of nutrients. The ability to identify factors limiting algal growth is of considerable importance to our understanding of the ecology of aquatic plants and to water management practices. Methods used to identify limiting resources in the past have included a) analysis of nutrient availability, b) elemental composition and cell quotas for various nutrients, c) bio-assays monitoring growth of test species or of natural populations following nutrient enrichment and d) measurements of various physiological parameters, such as enhancement of respiration and dark carbon fixation rates and perturbation of photosynthetic rate following re-supply of nutrients.¶In this paper we briefly review the merits and methodological limitations of these approaches for the assessment of the nutrient status of algal populations. We discuss how an understanding of biochemical and metabolic changes induced by nutrient limitation has led to the development of rapid and simple tools to monitor the nutrient status of aquatic plants and algae. In particular, we describe the use of transient changes in chlorophyll a fluorescence as a potential tool for rapid assessment of algal nutrient status and the development of molecular probes specific to nutrient limited cells, such as flavodoxin as a diagnostic tool for Fe-limitation.

Growth, photosynthesis, respiration, and intracellular free amino acid profiles in the unicellular alga Cyanidium caldarium. Effect of nutrient limitation and resupply

Growth, intrácellular free amino acid pools and photosynthetic and respiratory activities in nutrient sufficient cells and in N‐ K‐ and P‐limited cells of Cyanidium caldarium (Tilden) Geitler, and responses to nutrient resupply were investigated. Addition of ammonium to N‐limited cells and of phosphate to P‐limited cells resulted in a stimulation of dark respiration and in a decrease in photosynthetic oxygen evolution. Addition of K to K‐limited cells had no effect on rates of photosynthesis and respiration. Nutrient limited cells and sufficient cells exhibited different free amino acid profiles. Upon resupply of ammonium to N‐limited cells levels of glutamine, citrulline, arginine, alanine, and serine increased. Also the levels of δ‐aminolevulinic acid (δ‐ALA) and putrescine increased notably. On adding phosphate to P‐limited cells the level of glutamate decreased significantly whereas the level of alanine increased and the concentrations of other amino acids remained unaffected. On adding potassium to K‐limited cells there was an increase in glutamate and citrulline concentrations, and a decrease in putrescine concentration, whereas concentrations of arginine and alanine remained at the very high levels observed already before addition. Resuspension of N‐ and K‐limited cells in a complete growth only after 25‐30h. In P‐limited cells resumption of growth in complete medium occurred progressively and reached the maximum rate 30h later. P‐, K‐ and N‐ limited cells resuspended into sufficient media showed different rates of ammonium and phosphate assimilation. The pattern of recovery from nutrient limitation is discussed according to the cellular role fulfilled by the nutrient which was growth rate‐limiting.

Control of cell division in Paramecium tetraurelia: Effects of abrupt changes in nutrient level on accumulation of macronuclear DNA and cell mass

In the cell cycle of Paramecium there are three points of interaction between cell growth-related processes and the processes of macronuclear DNA replication and cell division: initiation of DNA synthesis, regulation of the rates of growth and DNA accumulation, and initiation of cell division. This study examines the regulation of the latter two processes by analysis of the response of each to abrupt changes in nutrient level brought about either by transferring dividing cells from a steady-state chemostat culture to medium with unlimited food, or by transferring well-fed dividing cells to exhausted medium. The rates of DNA accumulation and cell growth respond quickly to changes in nutrient level. The amounts of these cell components accumulated during the cell cycle following a shift in nutrient level are typical of those occurring during equilibrium growth under postshift conditions. Commitment to division occurs at a fixed interval prior to fission that is similar in well-fed and nutrient-limited cells. Initiation of cell division in Paramecium is associated with accumulation of a threshold DNA increment, whose level is largely independent of nutritive conditions. The amount of DNA accumulated during the cell cycle varies with nutritional conditions because the rates of growth and DNA accumulation are affected by nutrient level; slowly growing cells accumulated relatively little DNA during the fixed interval between commitment to cell division and fission.

A semicontinuous culture model that links cell growth to extracellular nutrient concentration

During semicontinuous culture, a sample of fixed volume is removed at regular time intervals to make measurements and/or harvest culture components, and an equal volume of fresh medium is immediately added to the culture, thereby instantaneously enhancing nutrient concentrations and diluting cell concentration. The resulting cell concentration versus time curve (i.e., the actual cell growth curve) has a saw-toothed appearance because of the periodic dilution of cell concentration. The observed cell concentrations correspond to the peaks of the saw-toothed curve. Cell growth rates are estimated from the locus of observed cell concentrations (i.e., from the apparent growth curve obtained by connecting the peaks of the saw-toothed curve). The sole preexisting model (Fencl's mode) for estimating cell growth rate is valid only when the cells are growing exponentially at a constant rate between samplings. This model has limited validity: despite the periodic enhancement of nutrient concentration, cell growth between samplings eventually causes nutrient depletion, and the cells cease to grow exponentially. Failure to recognize the limits of validity for Fencl' model has resulted in many erroneous applications of the model and, consequently, many incorrect estimates of cell growth rates. To provide a means for correctly estimating cell growth rates, Fencl's exponential model was extended, and a new model that describes the effects of nutrient depletion on cell growth in semi-continuous culture was obtained. The new model shows that exhaustion of a single growth-limiting nutrient in semicontinuous culture causes the locus of cell concentrations observed at time intervals of Deltat to follow a logistic growth curve. The actual cell growth rate was shown to equal the apparent logistic growth rate plus the effective dilution rate -Deltat(-1) In (1 - f), where f is the ratio of sample volume to total culture volume. Moreover, the model predicts that both the apparent logistic growth rate and the apparent steady-state cell concentration should rise linearly with the concentration of growth-limiting nutrient in the input medium, but fall linearly with increases in the effective dilution rate. The new logistic model for nutrient-limited cell growth in semicontinuous culture was successfully tested using published data for Asterionella formosa, Cyclotella meneghiniana, Daucus carota, and strain L mouse cells.

Effects of Nitrate and Phosphate Limitation on Cyclostat Growth of Two Freshwater Diatoms

The effects of N and P limitation on cell division and on cellular contents of limiting and non-limiting nutrients were examined in cyclostat cultures of the freshwater diatoms Asterionella formosa and Fragilaria crotonensis. Nutrient-limited cell cycles of both species were readily entrained by a 14 h/10 h light/dark cycle. In F. crotonensis, the maxima relative to the onset of the light phase of the photocycle occurred at approximately − 1 to +1 h for cell division, +11 to 13 h for cell N, and +21 to 23 h for cell P. In A. formosa the maxima occurred at approximately +3 to 10 h for cell division and +2 to 4 h for cell N and cell P. Changes in culture dilution rate (integrated growth) and in the type of nutrient limiting growth did not significantly alter these times for either species. The cell-number oscillations can be explained by a simple exponential function for the instantaneous population growth rate, which occurs after a single modal transition time τ, which is characteristic for each species. The consistency of phase relations between N and P limitations and culture dilution rates suggests that the temporal organization for events within the cell cycle of each organism is independent of the nutrient-limited status of the cell populations.

Marine diatoms grown in chemostats under silicate or ammonium limitation. III. Cellular chemical composition and morphology of Chaetoceros debilis, Skeletonema costatum, and Thalassiosira gravida

Three marine diatoms, Skeletonema costatum, Chaetoceros debilis, and Thalassiosira gravida were grown under no limitation and ammonium or silicate limitation or starvation. Changes in cell morphology were documented with photomicrographs of ammonium and silicate-limited and non-limited cells, and correlated with observed changes in chemical composition. Cultures grown under silicate starvation or limitation showed an increase in particulate carbon, nitrogen and phosporus and chlorophyll a per unit cell volume compared to non-limited cells; particulate silica per cell volume decreased. Si-starved cells were different from Si-limited cells in that the former contained more particulate carbon and silica per cell volume. The most sensitive indicator of silicate limitation or starvation was the ratio C:Si, being 3 to 5 times higher than the values for non-limited cells. The ratios Si:chlorophyll a and S:P were lower and N:Si was higher than non-limited cells by a factor of 2 to 3. The other ratios, C:N, C:P, C:chlorophyll a, N:chlorophyll a, P:chlorophyll a and N:P were considered not to be sensitive indicators of silicate limitation or starvation. Chlorophyll a, and particulate nitrogen per unit cell volume decreased under ammonium limitation and starvation. NH4-starved cells contained more chlorophyll a, carbon, nitrogen, silica, and phosphorus per cell volume than NH4-limited cells. N:Si was the most sensitive ratio to ammonium limitation or starvation, being 2 to 3 times lower than non-limited cells. Si:chlorophyll a, P:chlorophyll a and N:P were less sensitive, while the ratios C:N, C:chlorophyll a, N:chlorophyll a, C:Si, C:P and Si:P were the least sensitive. Limited cells had less of the limiting nutrient per unit cell volume than starved cells and more of the non-limiting nutrients (i.e., silica and phosphorus for NH4-limited cells). This suggests that nutrient-limited cells rather than nutrient-starved cells should be used along with non-limited cells to measure the full range of potential change in cellular chemical composition for one species under nutrient limitation.