Fast-growing Cells Research Articles

Promises and failures of gallium as an antibacterial agent.

Gallium has a long history as a diagnostic and chemotherapeutic agent. The pharmacological properties of Ga(III) rely on chemical mimicry; when Ga(III) is exogenously supplied to living cells it can replace Fe(III) within target molecules, thereby perturbing bacterial metabolism. Ga(III)-induced metabolic distresses are dramatic in fast-growing cells, like bacterial cells. Interest in the antibacterial properties of Ga(III) has been raised by the compelling need for novel drugs to combat multidrug-resistant bacteria and by the shortage of new antibiotic candidates in the pharmaceutical pipeline. Ga(III) activity has been demonstrated, both in vitro and in animal models of infections, on several bacterial pathogens, also including intracellular and biofilm-forming bacteria. Ga(III) activity is affected by iron availability and the metabolic state of the cell, being maximal in iron-poor media and in respiring cells. Synergism between Ga(III) and antibiotics holds promise as last resort therapy for infections sustained by pandrug-resistant bacteria.

Mitochondria-targeted Resveratrol Derivatives Act as Cytotoxic Pro-oxidants

Resveratrol derivatives bearing an O-linked mitochondria-targeting 4-triphenylphosphoniumbutyl group at either position 3 or position 4' are prooxidant and cytotoxic for cultured cells, selectively killing fast-growing cells when supplied in the low μM range. Resveratrol is essentially without effect under these experimental conditions, while the cytotoxicity of the mitochondriotropic derivatives increases if they are methylated on the remaining hydroxyls. Experiments with Bax(-/-)/Bak(-/-) cells and a pan-caspase inhibitor show that cell death is mostly of the necrotic type. Cytotoxicity is due to ROS produced upon accumulation of the compounds into mitochondria, and specifically to H2O2, since externally added membrane-permeant catalase largely prevents cell death while superoxide dismutase potentiates toxicity. The mitochondriotropic compounds cause ROS-independent depolarization of in situ mitochondria. Effectiveness is increased if resveratrol hydroxyls are acetylated or methylated; this excludes the involvement of autooxidation of the polyphenolic nucleus and a protonophoric cycle as the causes of ROS generation and of depolarization, respectively. Resveratrol-triphenylphosphonium conjugates may thus represent a new class of chemotherapeutic agents, redox-active "mitocans", whose mechanisms of action and in vivo activity are worthy of further investigation.

Elucidating Metabolic Variability in Isogenic Microbial Populations Arising due to Noise in Protein Expression

Members of a population of isogenic microbes vary in their protein expression states because of stochastic gene expression. Advances in single-cell proteomics on model organisms like E. coli and S. cerevisiae are uncovering this heterogeneity in protein copy numbers. Here we use genome-scale flux balance models to study the effect of heterogeneity in protein expression on metabolic behavior. We predict wide distribution in specific growth rates among the members of the population in accordance with recent single-cell growth rate measurements. Using flux balance analysis along with principal component analysis enables us to identify sub-populations which differ in their metabolic pathway usage. In case of E. coli grown in a minimal medium, we predict presence of slow-growing acetate secreting cells, fast-growing CO2 secreting cells and shifting preference between glycolysis and ED pathway to metabolize glucose. Preliminary population-level measurements support our prediction of acetate secreting cells in aerobic growth conditions. We also find that variability in expression of few genes may be sufficient to capture most of the metabolic variability of the entire population. We extend this study to S. cerevisiae for which the requisite data is becoming available.

Tumor-Targeting Transferrin Nanoparticles for Systemic Polymerized siRNA Delivery in Tumor-Bearing Mice

Transferrin (TF) is widely used as a tumor-targeting ligand for the delivery of anticancer drugs because the TF receptor is overexpressed on the surface of various fast-growing cancer cells. In this article, we report on TF nanoparticles as an siRNA delivery carrier for in vivo tumor-specific gene silencing. To produce siRNA carrying TF nanoparticles (NPs), both TF and siRNA were chemically modified with sulfhydryl groups that can build up self-cross-linked siRNA-TF NPs. Self-polymerized 5'-end thiol-modified siRNA (poly siRNA, psi) and thiolated transferrin (tTF) were spontaneously cross-linked to form stable NPs (psi-tTF NPs) under optimized conditions, and they could be reversibly degraded to release functional monomeric siRNA molecules under reductive conditions. Receptor-mediated endocytosis of TF induced rapid tumor-cell-specific uptake of the psi-tTF NPs, and the internalized NPs resulted in a downregulation of the target protein in red-fluorescent-protein-expressing melanoma cancer cells (RFP/B16F10) with negligible cytotoxicity. After systemic administration, the psi-tTF NPs showed marked accumulation at the tumor, leading to successful target-gene silencing in vivo. This psi-tTF NP system provided a safe and effective strategy for in vivo systemic siRNA delivery for cancer therapy.

Mechanisms and mechanics of cell competition in epithelia

When fast-growing cells are confronted with slow-growing cells in a mosaic tissue, the slow-growing cells are often progressively eliminated by apoptosis through a process known as cell competition. The underlying signalling pathways remain unknown, but recent findings have shown that cell crowding within an epithelium leads to the eviction of cells from the epithelial sheet. This suggests that mechanical forces could contribute to cell elimination during cell competition.

Heterogeneity in protein expression induces metabolic variability in a modeled Escherichia coli population

Stochastic gene expression can lead to phenotypic differences among cells even in isogenic populations growing under macroscopically identical conditions. Here, we apply flux balance analysis in investigating the effects of single-cell proteomics data on the metabolic behavior of an in silico Escherichia coli population. We use the latest metabolic reconstruction integrated with transcriptional regulatory data to model realistic cells growing in a glucose minimal medium under aerobic conditions. The modeled population exhibits a broad distribution of growth rates, and principal component analysis was used to identify well-defined subpopulations that differ in terms of their pathway use. The cells differentiate into slow-growing acetate-secreting cells and fast-growing CO2-secreting cells, and a large population growing at intermediate rates shift from glycolysis to Entner-Doudoroff pathway use. Constraints imposed by integrating regulatory data have a large impact on NADH oxidizing pathway use within the cell. Finally, we find that stochasticity in the expression of only a few genes may be sufficient to capture most of the metabolic variability of the entire population.

A dynamic model of the cellular carbon to chlorophyll ratio applied to a batch culture and a continental shelf ecosystem

A novel parameterization of the dynamical relationship between cellular carbon (C) and chlorophyll (Chl) is developed using a Chl synthesis term that includes the physiological status of the cell and the effect of packaging of pigments within cells. The geometric derivation highlights the non‐linear relationship between Chl content and absorption due to the package effect. When parameterized for a generic 3 µm radius phytoplankton cell, the model reproduces the magnitude and daily variations of C : Chl and C : nitrogen ratios of the diatom Skeletonema costatum in published laboratory experiments. The parameterization is then applied in a three‐dimensional biogeochemical model containing three phytoplankton classes in the coastal waters off southeast Tasmania, Australia, which demonstrates the behavior of the dynamic Chl parameterization over a range of light‐ and nutrient‐limiting environments for phytoplankton of different sizes and growth rates. The model produces C : Chl ratios of ∼ 12–20 (weight : weight) and ∼ 60–80 for phytoplankton communities dominated by fast‐growing small and fast‐growing large cells, respectively, close to the ratios of 17 and 76 observed at two sampling stations during periods with diatom‐ and flagellate‐dominated communities. Throughout the simulation, community C : Chl ratios generally vary between 12 and 200, which is similar to the range observed globally. In the new parameterization, C : Chl ratios are most influenced by the package effect for light‐limited, slow‐growing large microalgae, with physiological processes becoming important for smaller, nutrient‐limited, fast‐growing microalgae.

Age-Related Methylation Patterning of Housekeeping Genes and Tissue-Specific Genes is Distinct Between the Stomach Antrum and Body

The methylation-variable sites around CpG islands are frequently overmethylated in Helicobacter pylori-infected stomachs. Age-related patterns of the overmethylation changes were compared between the fast-growing antrum cells and the slow-growing body cells. A total of 316 H. pylori-positive tissues and 380 H. pylori-negative tissues were obtained by endoscopic biopsy. The methylation-variable sites of ten housekeeping genes and nine tissue-specific genes were semiquantitatively analyzed, based on the ten-level classification of methylation-specific PCR intensity. The overmethylated genes were scored when their methylation levels were higher than an intermediate level of each gene common in the H. pylori-negative mucosa. The age-dependent methylation level of the inactive APC gene observed similarly in the antrum and the body was used as an age standard of methylation variation in a biopsy tissue. The overmethylation of housekeeping genes and stomach-specific genes rapidly increased to a high plateau frequency in the young-aged APC methylation cases (mean age: 43 years) in the H. pylori-positive antrum. In the H. pylori-positive body, most of the overmethylated housekeeping genes slowly increased to a peak frequency in the middle-aged APC methylation cases (mean age: 53 years). The housekeeping gene pairs showed high correlations (Spearman's correlation coefficient > 0.4) in both the antrum and the body. The overmethylation of housekeeping genes rapidly and slowly increased to a high frequency in concordance with a rapid and slow growth of epithelial cells in the H. pylori-infected stomach.

FtsH-Mediated Coordination of Lipopolysaccharide Biosynthesis in Escherichia coli Correlates with the Growth Rate and the Alarmone (p)ppGpp

The outer membrane is the first line of defense for Gram-negative bacteria and serves as a major barrier for antibiotics and other harmful substances. The biosynthesis of lipopolysaccharides (LPS), the essential component of the outer membrane, must be tightly controlled as both too much and too little LPS are toxic. In Escherichia coli, the cellular level of the key enzyme LpxC, which catalyzes the first committed step in LPS biosynthesis, is adjusted by proteolysis carried out by the essential and membrane-bound protease FtsH. Here, we demonstrate that LpxC is degraded in a growth rate-dependent manner with half-lives between 4 min and >2 h. According to the cellular demand for LPS biosynthesis, LpxC is degraded during slow growth but stabilized when cells grow rapidly. Disturbing the balance between LPS and phospholipid biosynthesis in favor of phospholipid production in an E. coli strain encoding a hyperactive FabZ protein abolishes growth rate dependency of LpxC proteolysis. Lack of the alternative sigma factor RpoS or inorganic polyphosphates, which are known to mediate growth rate-dependent gene regulation in E. coli, did not affect proteolysis of LpxC. In contrast, absence of RelA and SpoT, which synthesize the alarmone (p)ppGpp, deregulated LpxC degradation resulting in rapid proteolysis in fast-growing cells and stabilization during slow growth. Our data provide new insights into the essential control of LPS biosynthesis in E. coli.

Biodiversity Increases the Productivity and Stability of Phytoplankton Communities

Global biodiversity losses provide an immediate impetus to elucidate the relationships between biodiversity, productivity and stability. In this study, we quantified the effects of species richness and species combination on the productivity and stability of phytoplankton communities subject to predation by a single rotifer species. We also tested one mechanism of the insurance hypothesis: whether large, slow-growing, potentially-defended cells would compensate for the loss of small, fast-growing, poorly-defended cells after predation. There were significant effects of species richness and species combination on the productivity, relative yield, and stability of phytoplankton cultures, but the relative importance of species richness and combination varied with the response variables. Species combination drove patterns of productivity, whereas species richness was more important for stability. Polycultures containing the most productive single species, Dunaliella, were consistently the most productive. Yet, the most species rich cultures were the most stable, having low temporal variability in measures of biomass. Polycultures recovered from short-term negative grazing effects, but this recovery was not due to the compensation of large, slow-growing cells for the loss of small, fast-growing cells. Instead, polyculture recovery was the result of reduced rotifer grazing rates and persisting small species within the polycultures. Therefore, although an insurance effect in polycultures was found, this effect was indirect and unrelated to grazing tolerance. We hypothesize that diverse phytoplankton assemblages interfered with efficient rotifer grazing and that this “interference effect” facilitated the recovery of the most productive species, Dunaliella. In summary, we demonstrate that both species composition and species richness are important in driving patterns of productivity and stability, respectively, and that stability in biodiverse communities can result from an alteration in consumer functioning. Our findings underscore the importance of predator-prey dynamics in determining the relationships between biodiversity, productivity and stability in producer communities.

Euphorbia mauritanica and Kedrostis hirtella extracts can induce anti-proliferative activities in lung cancer cells

Cancer is a public health problem in the world accounting for most of the deaths. Currently, common treatment of cancer such as chemotherapy works by killing fast-growing cancer cells. Unfortunately, chemotherapy cannot tell the difference between cancer cells and fast-growing healthy cells, including red and white blood cells. As a result, one of the most serious potential side effects of some types of chemotherapy is a low white blood cell count that makes it unreliable (Parkin et al. [34]; Pauk et al. [3]). Even though intense research has been going on in recent years, successful therapeutic targets against this disease have been elusive. In this study, we evaluate the anti-proliferative activity of Euphorbia mauritanica and Kedrostis hirtella in lung cancer. In our assessment it was observed that E. mauritanica and K. hirtella were able to induce cell death at 5 μg/ml in A549 cells over 22 h and at 10 μg/ml over 24 h in the Lqr1 cell line. Molecular analysis of DNA fragmentation and Annexin V were used to examine the type of cell death induced by E. mauritanica and K. hirtella extracts. These results showed an increase in necrotic and apoptotic characteristics with both nuclear DNA fragmentation and smear. Therefore, these results suggest that E. mauritanica and K. hirtella may play a role in inducing cell death in lung cancer cells. However, further studies need to be conducted to ascertain these results.

Discovery of a Plasmodium falciparum Glucose-6-phosphate Dehydrogenase 6-phosphogluconolactonase Inhibitor (R,Z)-N-((1-Ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (ML276) That Reduces Parasite Growth in Vitro

A high-throughput screen of the NIH's MLSMR collection of ∼340000 compounds was undertaken to identify compounds that inhibit Plasmodium falciparum glucose-6-phosphate dehydrogenase (PfG6PD). PfG6PD is important for proliferating and propagating P. falciparum and differs structurally and mechanistically from the human orthologue. The reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PD) is the first, rate-limiting step in the pentose phosphate pathway (PPP), a key metabolic pathway sustaining anabolic needs in reductive equivalents and synthetic materials in fast-growing cells. In P. falciparum , the bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (PfGluPho) catalyzes the first two steps of the PPP. Because P. falciparum and infected host red blood cells rely on accelerated glucose flux, they depend on the G6PD activity of PfGluPho. The lead compound identified from this effort, (R,Z)-N-((1-ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide, 11 (ML276), is a submicromolar inhibitor of PfG6PD (IC(50) = 889 nM). It is completely selective for the enzyme's human isoform, displays micromolar potency (IC(50) = 2.6 μM) against P. falciparum in culture, and has good drug-like properties, including high solubility and moderate microsomal stability. Studies testing the potential advantage of inhibiting PfG6PD in vivo are in progress.

The P2X7 receptor is a key modulator of aerobic glycolysis

Ability to adapt to conditions of limited nutrient supply requires a reorganization of the metabolic pathways to balance energy generation and production of biosynthetic intermediates. Several fast-growing cells overexpress the P2X7 receptor (P2X7R) for extracellular ATP. A feature of this receptor is to allow growth in the absence of serum. We show here that transfection of P2X7R allows proliferation of P2X7R-transfected HEK293 (HEK293-P2X7) cells not only in the absence of serum but also in low (4 mM) glucose, and increases lactate output compared with mock-transfected HEK293 (HEK293-mock) cells. In HEK293-P2X7, lactate output is further stimulated upon addition of exogenous ATP or the mitochondrial uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). In the human neuroblastoma cell line ACN, lactate output is also dependent on P2X7R function. P2X7R-expressing cells upregulate (a) the glucose transporter Glut1, (b) the glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (G3PDH), (c) phosphofructokinase (PFK), (d) pyruvate kinase M2 (PKM2) and (e) pyruvate dehydrogenase kinase 1 (PDHK1); furthermore, P2X7R expression (a) inhibits pyruvate dehydrogenase (PDH) activity, (b) increases phosphorylated Akt/PKB and hypoxia-inducible factor 1α (HIF-1α) expression and (c) enhances intracellular glycogen stores. In HEK293-P2X7 cells, glucose deprivation increases lactate production, expression of glycolytic enzymes and ph-Akt/PKB level. These data show that the P2X7R has an intrinsic ability to reprogram cell metabolism to meet the needs imposed by adverse environmental conditions.

Iron fertilization and the structure of planktonic communities in high nutrient regions of the Southern Ocean

In this review article, plankton community structure observations are analyzed both for artificial iron fertilization experiments and also for experiments dedicated to the study of naturally iron-fertilized systems in the Atlantic, Indian and Pacific sectors of the Southern Ocean in the POOZ (Permanently Open Ocean Zone) and the PFZ (Polar Frontal Zone). Observations made in natural systems are combined with those from artificially perturbed systems, in order to evaluate the seasonal evolution of pelagic communities, taking into account controlling factors related to the life cycles and the ecophysiology of dominant organisms. The analysis considers several types of planktonic communities, including both autotrophs and heterotrophs. These communities are spatially segregated owing to different life strategies. A conceptual general scheme is proposed to account for these observations and their variability, regardless of experiment type. Diatoms can be separated into 2 groups: Group 1 has slightly silicified fast growing cells that are homogeneously distributed in the surface mixed layer, and Group 2 has strongly silicified slowly growing cells within discrete layers. During the growth season, Group 1 diatoms show a typical seasonal succession of dominant species, within time windows of development that are conditioned by physical factors (light and temperature) as well as endogenous specific rhythms (internal clock), and biomass accumulation is controlled by the availability of nutrients. Group 1 diatoms are not directly grazed by mesozooplankton which is fed by protozooplankton, linking the microbial food web to higher trophic levels. Instead, successive dominant species of Group 1 are degraded via bacterial activity at the end of their growth season. Organic detritus fragments feed protozooplankton and mesozooplankton. The effective silicon pump leads to the progressive disappearance of silicic acid in surface waters. In contrast, Group 2 is resistant to grazing due to its strong silicification, and its biomass accumulates continuously but relatively slowly throughout the productive period. Group 2 diatoms are concentrated at or near the seasonal pycnocline and thus benefit from upward nutrient fluxes by diapycnal mixing. The decrease in light and the deep convective mixing in the fall produce both light and nutrient limitation leading to a massive carbon export of Group 2 diatoms, a major annual event of the biological pump. This scheme describes the seasonal evolution of plankton communities in surface waters of the Southern Ocean. The scheme could probably be extended to ecosystems that are characterized by a seasonal bloom under influence of iron or other nutrients.

Nanoparticles for Targeted Delivery of Active Agents against Tumor Cells

Cancer therapy may still be considered as one of the most challenging issues in pharmaceutical sciences. Various new technologies and molecules have been developed to meet the needs of this devastating disease, but there is a long way to go. One of the most important challenges is finding a drug molecule or drug delivery system which only influences the cancer cells. This has not been achieved so far even though a great deal of research has focused on this goal. Most of the cytotoxic agents used for cancer treatment are toxic in nature and not only have an effect on tumor cells, but also normal cells are damaged when exposed with them. As a result, patients may experience intensive side effects which may lead to an early termination in chemotherapy due to their severity. Targeting the cancer cells specifically and selectively seems to be the best solution for this obstacle. Targeting the cancer cells occurs via two different strategies: passive targeting and active targeting. The conventional approach used for cancer therapy may be considered a sort of passive targeting. Cytotoxic agents used for cancer treatment select the cancer cells due to their faster growth rate. Inevitably, normal fast growing cells and normal fast dividing cells may damage following conventional chemotherapy. In 1986, Matsumura and Maeda brought up a new concept in passive targeting called enhanced permeation and retention (EPR) effect which was widely used in many researches as an approach for targeting tumor cells. In this strategy, the drug delivery system benefits from defective vasculature, fenestrations, and poor lymphatic drainage in tumor tissues. Utilizing molecular targeting moieties is another strategy which is widely used in new researches for targeting tumor cells actively and diminishing chemotherapy side effects. This special issue will primarily address new approaches for passive and active targeted cancer therapy with a focus on nanoparticle formulations specially those ones that specifically and selectively target tumors. There is a peper which describes using a natural protein, atelocollagen for oligonucleotide delivery, and evaluating its biological function. The researchers found that when oligonucleotides were used concomitantly with atelocollagen, paracellular flux was enhanced, but when each of these two constituents of the formulation was used separately, no effect was observed. Another paper reviews poly(lactic-co-glycolic acid)- (PLGA-) based nanoparticles especially echogenic ones to enhance gene delivery. Ultrasound may be used to enhance localized gene delivery in tumors. On the other hand, nanoparticles themselves may utilize targeting mechanisms to enhance the targeting potential of the current system. Another paper describes microemulsions in the form of nanocarriers for passive tumor targeting. In this study, an experimental design layout was used to optimize the excipients selection for microemulsion nanocarriers. These nanocarriers possess the ability of solubilizing hydrophobic drugs and physical stability alongside with their capability of passive targeting. In another paper human serum albumin was used as a naturally occurring protein to create nanoparticles for enhanced drug delivery in breast cancer. Albumin-based nanoparticles were further cationized by polyethylenimine. It was shown that delivery efficacy was improved by passive EPR strategy. One of the papers also reviews carbon nanotubes for cancer therapy and designing drug delivery systems. Carbon nanotubes present a great canvas for surface engineering and creating novel targeted nanoparticles. Another paper presents glucan particles which actively target cancer cells. Glucan particles which benefit from β-glucan derived from the cell wall of Saccharomyces cerevisiae provide receptor-mediated uptake by phagocytic macrophage cells who express β-glucan receptors. Different types of nanoparticles were incorporated or surface bounded to these glucan particles to deliver doxorubicin to macrophages. Macrophages further migrate into solid tumors carrying the cytotoxic agent to the target tissues like a Trojan horse, while they are resistant to the effects of doxorubicin due to their nondividing differentiated nature. Another paper reviews noble metal nanoparticles with a wide variety of biomedical applications which are used for both therapeutic and diagnostic purposes. They also present facile surface chemistry and the possibility of functionalizing on their surfaces. Finally, in one of the papers, a lipoplatin formulation consisting of liposomal encapsulated cisplatin is reviewed. The purpose of this formulation is to become a substitute for the original cisplatin and has gained positive results in clinical trials. Lipoplatin gives 200-fold higher concentrations in tumor tissues in comparison with cisplatin once more because of the compromised endothelium of tumor tissues. To sum up, this special issue tries to provide the readers some insight about the targeted nanoparticles which are designed for cancer therapy both for small molecular drugs and gene delivery systems. Rassoul Dinarvand Paulo Cesar de Morais Antony D'Emanuele

Proteomic and metabolomic characterization of CHO DP-12 cell lines with different high passage histories

Background For industrial pharmaceutical protein production fast growing, high producing and robust cell lines are required. To select more pH shift permissive and fast growing sub-populations, the CHO DP-12 (ATCC clone #1934) cell line, an anti-IL8 antibody producing CHO K1 (DHFR) clone, was continuously subcultured at high viability (>90 %) for more than four hundred days in shaking flasks using a chemically defined medium. During this long-term cultivation there was a repeated shift in pH and most robust and fast growing cells became accumulated [1]. Cell samples were cryopreserved at four different time points, after 21, 95, 165 and 420 days (in the following named sub-populations (SP)). The effects of long-term passaging before cryopreservation correlating with an increase in specific growth rate as well as changes in product formation and metabolism were examined in parallel bench-top bioreactor cultivation of SP21, SP95, SP165 and SP420 sub-populations. During exponential growth phase samples were taken for the analyses of differences in intracellular metabolites and protein expression (Please consider article “Growth characterization of CHO DP-12 cell lines with different high-passage histories” by Heinrich et al. in this issue for a detailed discussion of long-term cultivation, changes in specific growth rate, product formation and metabolic shifts).

Growth Rate-Dependent Control in Enterococcus faecalis: Effects on the Transcriptome and Proteome, and Strong Regulation of Lactate Dehydrogenase

Enterococcus faecalis V583 was grown in a glucose-limited chemostat at three different growth rates (0.05, 0.15, and 0.4 h⁻¹). The fermentation pattern changed with growth rate, from a mostly homolactic profile at a high growth rate to a fermentation dominated by formate, acetate, and ethanol production at a low growth rate. A number of amino acids were consumed at the lower growth rates but not by fast-growing cells. The change in metabolic profile was caused mainly by decreased flux through lactate dehydrogenase. The transcription of ldh-1, encoding the principal lactate dehydrogenase, showed very strong growth rate dependence and differed by three orders of magnitude between the highest and the lowest growth rates. Despite the increase in ldh-1 transcript, the content of the Ldh-1 protein was the same under all conditions. Using microarrays and quantitative PCR, the levels of 227 gene transcripts were found to be affected by the growth rate, and 56 differentially expressed proteins were found by proteomic analyses. Few genes or proteins showed a growth rate-dependent increase or decrease in expression across the whole range of conditions, and many showed a maximum or minimum at the middle growth rate (i.e., 0.15 h⁻¹). For many gene products, a discrepancy between transcriptomic and proteomic data were seen, indicating posttranscriptional regulation of expression.

Essential Gene Pathways for Glioblastoma Stem Cells: Clinical Implications for Prevention of Tumor Recurrence

Glioblastoma (World Health Organization/WHO grade IV) is the most common and most aggressive adult glial tumor. Patients with glioblastoma, despite being treated with gross total resection and post-operative radiation/chemotherapy, will almost always develop tumor recurrence. Glioblastoma stem cells (GSC), a minor subpopulation within the tumor mass, have been recently characterized as tumor-initiating cells and hypothesized to be responsible for post-treatment recurrence because of their enhanced radio-/chemo-resistant phenotype and ability to reconstitute tumors in mouse brains. Genome-wide expression profile analysis uncovered molecular properties of GSC distinct from their differentiated, proliferative progeny that comprise the majority of the tumor mass. In contrast to the hyperproliferative and hyperangiogenic phenotype of glioblastoma tumors, GSC possess neuroectodermal properties and express genes associated with neural stem cells, radial glial cells, and neural crest cells, as well as portray a migratory, quiescent, and undifferentiated phenotype. Thus, cell cycle-targeted radio-chemotherapy, which aims to kill fast-growing tumor cells, may not completely eliminate glioblastoma tumors. To prevent tumor recurrence, a strategy targeting essential gene pathways of GSC must be identified and incorporated into the standard treatment regimen. Identifying intrinsic and extrinsic cues by which GSC maintain stemness properties and sustain both tumorigenesis and anti-apoptotic features may provide new insights into potentially curative strategies for treating brain cancers.

Cell-Cycle-Dependent Active Thermal Bystander Effect (ATBE)

To determine whether the active thermal bystander effect (ATBE) is cell-cycle dependent. Dividing cells were directly heated for 10 minutes and co-cultured for 24 hours with different bystander cells. We compared the ATBE for dividing and non-dividing human fibroblasts, as well as for dividing human white preadipocytes (HWP) and non-dividing, mature differentiated human white adipocytes. ATBE was assessed as loss of cell viability of the bystander cells by using the MTT assay. Cell-cycle analysis was performed by using flow cytometry. Dividing fibroblasts and preadipocytes showed a significant ATBE (P < 0.008 and P < 0.05) with loss of cell viability of about 10% in the temperature range of 40-48°C. There was no significant difference between the extent of the bystander effect for these two cell populations (P = 0.30). In contrast, non-dividing fibroblasts and mature adipocytes did not generate any ATBE within this temperature range. There was a statistically significant difference in ATBE between dividing and non-dividing cell subpopulations for both fibroblasts (P = 0.003) and preadipocytes (P < 0.001) compared to their non-dividing counterparts. These results suggest that the ATBE is a cell-cycle-dependent process which requires actively dividing cells as receiving bystander cells. The cell-cycle dependency of the ATBE could have useful clinical applications in selectively targeting fast growing cells such as tumor cells. Whether the yield of the ATBE can be amplified by synchronizing the exposure to the ATBE with specific phases of the cell cycle remains subject to further investigation.

LINKING TIME-DEPENDENT CARBON-FIXATION EFFICIENCIES IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE) TO UNDERLYING METABOLIC PATHWAYS(1).

The chl-specific short-term (14) C-based production (P(b) ) measurement is a widely used tool to understand phytoplankton responses to environmental stresses. However, among the metabolic consequences of these stresses is variability in lifetimes of newly fixed carbon that cause P(b) to range between chl-specific net primary production (NPP*) and chl-specific gross photosynthetic electron flow that is available for carbon reduction () depending on growth rate. To investigate the basis for this discrepancy, photosynthate utilization was characterized in Dunaliella tertiolecta Butcher grown at three different growth rates in N-limited chemostats. P(b) was measured throughout a 2 min to 24 h time course and showed clear growth-rate-dependent differences in lifetimes of newly fixed carbon. (14) C pulse-chase experiments revealed differences in patterns of carbon utilization between growth rates. At high growth rate, the majority of (14) C was initially fixed into polysaccharide and lipid, but the relative contribution of each labeled biochemical pool to the total label changed over 24 h. In fast-growing cells, labeled polysaccharides decreased 50%, while labeled lipids increased over the first 4 h. At low growth rate, (14) C was initially incorporated primarily into protein, but the contribution of labeled protein to the total label increased over the next 24 h. Together, time-resolved measurements of P(b) and cellular NAD and NADP content suggest an enhanced role for alternative dissipation pathways at very low growth rate. Findings of this study contribute to an integrated understanding of growth-rate-dependent shifts in metabolic processes from photosynthesis to net growth.