Decrease In Cell Yield Research Articles

LC-MS proteomic profiling of Caco-2 human intestinal cells exposed to the copper-chelating agent, triethylenetetramine: A preliminary study

Homeostasis of metal micronutrients such as copper is tightly regulated to ensure deficiency does not occur while restricting damage resulting from excess accumulation. Using LC-MS the effect on the proteome of intestinal Caco-2 cells of exposure to the chelator triethylenetetramine (TETA) was investigated. Continuous exposure of TETA at 25 μM to Caco-2 cells caused decreased cell yields and morphological changes. These effects were reversed when cells were no longer exposed to TETA.Quantitative proteomic analysis identified 957 mostly low-fold differentially expressed proteins, 41 of these returned towards control Caco-2 expression following recovery. Proteins exhibiting this “reciprocal” behaviour included upregulated deoxyhypusine hydroxylase (DOHH, 15.69- fold), a protein essential for eIF-5A factor hypsuination, a post translational modification responsible for eIF-5A maturation, which in turn is responsible for translation elongation. Exposure to TETA also resulted in 87 proteins, the expression of which was stable and remained differentially expressed following recovery.This study helps to elucidate the stable and transient proteomic effects of TETA exposure in intestinal cells.

Copper-induced non-monotonic dose response in Caco-2 cells.

Copper is an essential dietary micronutrient in humans for proper cell function; however, in excess, it is toxic. The human cell line Caco-2 is popular as an in vitro model for intestinal absorption and toxicology. This study investigated the response of exponentially growing Caco-2 cells to prolonged copper exposure (120h). An unexpected non-monotonic dose-response profile was observed in Caco-2 cells. Exposure to media supplemented with 3.125μM CuSO4 resulted in decreased cell yield vs. untreated. However, toxicity was progressively reduced from 90% at 3.125μM to 60% at 25μM. This effect was documented between 48 and 120h continuous exposure (p < 0.05). This triphasic toxicity curve was observed to be specific to copper in Caco-2 cells, as iron, manganese and zinc displayed monotonic dose-response profiles. Two inorganic copper forms, copper sulphate and copper chloride, were shown to conserve the non-monotonic dose-response curve. The triphasic effect was shown to be specific to Caco-2 cells. These results have implications for research investigating the effect of copper and other micronutrients using Caco-2 cells.

Spathaspora passalidarum selected for resistance to AFEX hydrolysate shows decreased cell yield.

This study employed cell recycling, batch adaptation, cell mating and high-throughput screening to select adapted Spathaspora passalidarum strains with improved fermentative ability. The most promising candidate YK208-E11 (E11) showed a 3-fold increase in specific fermentation rate compared to the parental strain and an ethanol yield greater than 0.45 g/g substrate while co-utilizing cellobiose, glucose and xylose. Further characterization showed that strain E11 also makes 40% less biomass compared to the parental strain when cultivated in rich media under aerobic conditions. A tetrazolium agar overlay assay in the presence of respiration inhibitors, including rotenone, antimycin A, KCN and salicylhydroxamic acid elucidated the nature of the mutational events. Results indicated that E11 has a deficiency in its respiration system that could contribute to its low cell yield. Strain E11 was subjected to whole genome sequencing and an ∼11 kb deletion was identified; the open reading frames absent in strain E11 code for proteins with predicted functions in respiration, cell division and the actin cytoskeleton, and may contribute to the observed physiology of the adapted strain. Results of the tetrazolium overlay also suggest that cultivation on xylose affects the respiration capacity in the wild-type strain, which could account for its faster fermentation of xylose as compared to glucose. These results support our previous finding that S. passalidarum has highly unusual physiological responses to xylose under oxygen limitation.

Clostridium thermocellum DSM 1313 transcriptional responses to redox perturbation.

BackgroundClostridium thermocellum is a promising consolidated bioprocessing candidate organism capable of directly converting lignocellulosic biomass to ethanol. Current ethanol yields, productivities, and growth inhibitions are industrial deployment impediments for commodity fuel production by this bacterium. Redox imbalance under certain conditions and in engineered strains may contribute to incomplete substrate utilization and may direct fermentation products to undesirable overflow metabolites. Towards a better understanding of redox metabolism in C. thermocellum, we established continuous growth conditions and analyzed global gene expression during addition of two stress chemicals (methyl viologen and hydrogen peroxide) which changed the fermentation redox potential.ResultsThe addition of methyl viologen to C. thermocellum DSM 1313 chemostat cultures caused an increase in ethanol and lactate yields. A lower fermenter redox potential was observed in response to methyl viologen exposure, which correlated with a decrease in cell yield and significant differential expression of 123 genes (log2 > 1.5 or log2 < −1.5, with a 5 % false discovery rate). Expression levels decreased in four main redox-active systems during methyl viologen exposure; the [NiFe] hydrogenase, sulfate transport and metabolism, ammonia assimilation (GS-GOGAT), and porphyrin/siroheme biosynthesis. Genes encoding sulfate transport and reduction and porphyrin/siroheme biosynthesis are co-located immediately downstream of a putative iscR regulatory gene, which may be a cis-regulatory element controlling expression of these genes. Other genes showing differential expression during methyl viologen exposure included transporters and transposases.ConclusionsThe differential expression results from this study support a role for C. thermocellum genes for sulfate transport/reduction, glutamate synthase-glutamine synthetase (the GS-GOGAT system), and porphyrin biosynthesis being involved in redox metabolism and homeostasis. This global profiling study provides gene targets for future studies to elucidate the relative contributions of prospective pathways for co-factor pool re-oxidation and C. thermocellum redox homeostasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0394-9) contains supplementary material, which is available to authorized users.

PTH-278 Aspirin enhances the response to radiation in colorectal cancer cell lines

Introduction Advances in surgical resection, neo-adjuvant therapies and efforts to ensure early detection have led to a reduction in colorectal cancer mortality rates since the 1970s. Despite this, around 40% of patients die from recurrence or metastatic disease, making it the second most common cause of cancer related death in the UK. Poor response to adjuvant treatment for colorectal cancer continues to pose a significant problem. Colorectal Cancer Stem Cells (CSC), are thought to be responsible for fuelling resistance and driving recurrent disease.1The identification of a reliable CSC marker, LGR5, 2has enabled further investigation and it has been demonstrated that both the function and survival of colorectal CSCs is driven by COX-2/PGE2and BCL-3/NFκB signalling pathways.3 Therefore, we aimed to determine whether Aspirin, through inhibiting PGE2production and BCL-3 expression, can enhance the response to radiotherapy, in colorectal cancer cells. Method Colorectal carcinoma (HCA7/P, LS174T, LoVo) and adenoma (AN/C1, RG/C2) cell lines were treated with Aspirin, (1–2 mM, 24–96 h). Cell survival (cell yield and apoptosis) and protein expression (analysed by Western blotting), were determined. Colorectal cancer cells were seeded at low densities and treated with combinations of Aspirin and/or γ-radiation (Cs-137 source). Cell survival was determined by crystal violet cell viability assay. Results 2 mM Aspirin decreased cell yield (e.g. LoVo- Control 26.7 × 106vs. 2 mM Aspirin 9.4 × 106, p = 0.002) and increased apoptosis (e.g. Lovo- Control 7.8 × 106vs. 2 mM Aspriin 19.5 × 106, p = 0.014) in carcinoma and adenoma cell lines at 72 and 96 h. Western analysis showed Aspirin treatment reduced the expression of CSC related markers in both colorectal carcinoma and adenoma cell lines. 2mM Aspirin increased the sensitivity to 2.5Gy radiation in colorectal carcinoma cells, leading to an approximate two-fold decrease in cell survival, (e.g. HCA7/P- 2.5Gy radiation 81.9% survival vs. 43.4% for 2.5Gy and 2 mM Aspirin combined). Suppression of BCL-3 expression, in addition to Aspirin, further enhanced response to radiation. Conclusion Aspirin decreases cell survival and increases apoptosis in colorectal carcinoma and adenoma cell lines. Furthermore, it enhances the response to radiation, reducing colorectal cancer cell survival. BCL-3 suppression may modulate this response. Clinical translation, to further explore Aspirin’s role as a novel adjunct to radiation, is underway in our multi-centre prospective cohort study, (ASPIRE). Disclosure of interest None Declared. References O’Brien CA. Nature 2007;445(7123):106–110 Barker N. Nature 2007;449(7165):1003–7 Al-Kharusi MRA. Carcinogenesis 2013;34(5):1150–1157

Different roles of two transcription factor B proteins in the hyperthermophilic archaeon Thermococcus kodakarensis

Two genes, TK1280 and TK2287, encode orthologous transcription factor B proteins (TFB1 and TFB2, respectively) in the hyperthermophilic archaeon Thermococcus kodakarensis. The functional difference between their TFBs remains unknown. While TFB1 and TFB2 displayed equivalent thermostability, mRNA levels of tfb1 at 93 °C were eightfold higher than those at 60 or 85 °C, and were 4- to 10-fold greater than those of tfb2 at all temperatures. This suggests that TFB1 is the abundant TFB in T. kodakarensis and is heat-inducible. By contrast, the mRNA level of tfb2 increased at 93 °C, but the levels were less than twofold of those at 60 or 85 °C. No significant differences in growth were observed among the DTF1 (∆tfb1, ∆pyrF), DTF2 (∆tfb2 ∆pyrF), and parental host strain KU216 (∆pyrF) at 60 °C. However, DTF2 showed a decrease in cell yield at 85 °C, and both DTF1 and DTF2 showed growth defects at 93 °C. Comparative transcriptome analysis between KU216 and DTF1 or DTF2 indicated that TFB1 apparently controls the expression of genes essential for motility/adhesion, whereas TFB2 regulates genes involved in mevalonate/lipid biosynthesis. In DTF1, the ratio of cells with flagella decreased at 85 and 93 °C, and reporter studies indicated that flaB1 transcription is dependent on TFB1 at 85 °C but not at 60 °C.

Dockerin-containing protease inhibitor protects key cellulosomal cellulases from proteolysis inClostridium cellulolyticum

Cellulosomes are key for lignocellulosic biomass degradation in cellulolytic Clostridia. Better understanding of the mechanism of cellulosome regulation would allow us to improve lignocellulose hydrolysis. It is hypothesized that cellulosomal protease inhibitors would regulate cellulosome architecture and then lignocellulose hydrolysis. Here, a dockerin-containing protease inhibitor gene (dpi) in Clostridium cellulolyticum H10 was characterized by mutagenesis and physiological analyses. The dpi mutant had a decreased cell yield on glucose, cellulose and xylan, lower cellulose utilization efficiency, and a 70% and 52% decrease of the key cellulosomal components, Cel48F and Cel9E respectively. The decreased cellulolysis is caused by the proteolysis of major cellulosomal components, such as Cel48F and Cel9E. Disruption of cel9E severely impaired cell growth on cellulose while loss of cel48F completely abolished cellulolytic activity. These observations are due to the combinational results of gene inactivation and polar effects caused by intron insertion. Purified recombinant Dpi showed inhibitory activity against cysteine protease. Taken together, Dpi protects key cellulosomal cellulases from proteolysis in H10. This study identified the physiological importance of cellulosome-localized protease inhibitors in Clostridia.

Laboratory Evolution and Multi-platform Genome Re-sequencing of the Cellulolytic Actinobacterium Thermobifida fusca

Biological utilization of cellulose is a complex process involving the coordinated expression of different cellulases, often in a synergistic manner. One possible means of inducing an organism-level change in cellulase activity is to use laboratory adaptive evolution. In this study, evolved strains of the cellulolytic actinobacterium, Thermobifida fusca, were generated for two different scenarios: continuous exposure to cellobiose (strain muC) or alternating exposure to cellobiose and glucose (strain muS). These environmental conditions produced a phenotype specialized for growth on cellobiose (muC) and an adaptable, generalist phenotype (muS). Characterization of cellular phenotypes and whole genome re-sequencing were conducted for both the muC and muS strains. Phenotypically, the muC strain showed decreased cell yield over the course of evolution concurrent with decreased cellulase activity, increased intracellular ATP concentrations, and higher end-product secretions. The muS strain increased its cell yield for growth on glucose and exhibited a more generalist phenotype with higher cellulase activity and growth capabilities on different substrates. Whole genome re-sequencing identified 48 errors in the reference genome and 18 and 14 point mutations in the muC and muS strains, respectively. Among these mutations, the site mutation of Tfu_1867 was found to contribute the specialist phenotype and the site mutation of Tfu_0423 was found to contribute the generalist phenotype. By conducting and characterizing evolution experiments on Thermobifida fusca, we were able to show that evolutionary changes balance ATP energetic considerations with cellulase activity. Increased cellulase activity is achieved in stress environments (switching carbon sources), otherwise cellulase activity is minimized to conserve ATP.

Critical microcarrier properties affecting the expansion of undifferentiated human embryonic stem cells

A variety of microcarriers may be used for the expansion of human embryonic stem cells (hESC) for cell therapy applications. This study investigated the effects of 10 types of microcarriers on hESC attachment efficiency, growth and pluripotency. High attachment efficiency was observed on uncoated microcarriers, however poor cell growth and/or gradual loss of pluripotency occurred during continuous passaging. Coating of the microcarriers with Matrigel resulted in higher cell yields and stable pluripotent states for at least three passages. Positively charged cylindrical cellulose microcarriers (DE52, DE53 and QA52) and large (190 μm) positively charged spherical microcarriers (Cytodex 1) exhibited high cell expansion potential and levels of pluripotency. Lower cell yields were obtained using smaller diameter spherical (65 μm and 10 μm) or macroporous beads. Instead of Matrigel, laminin coated microcarriers (DE53 and Cytodex 1) are capable of supporting the long term propagation and pluripotency of HES-2 and HES-3 cell lines. HES-2 cell line which was shown earlier to be shear resistant achieved similar cell growth and expression of pluripotent markers when cultured on both Matrigel (84% Tra-1-60, 1.43 × 10 6 cells/ml) and laminin (74% Tra-1-60, 1.37 × 10 6 cells/ml) coated microcarriers in spinner flasks. In contrast, HES-3 exhibited a decrease in cell yield, viability and pluripotent markers on laminin as compared with Matrigel coated microcarriers possibly due to shear sensitivity. Conventional microcarriers intended for propagation of mammalian cells are not suitable for long term propagation of hESC. Matrigel or laminin coating is essential for stable long term propagation of hESC on a variety of microcarriers.

Abstract 3856: Loss fo N or K-Ras B does not mimic the effects of farnesy transferase inhibited osteosarcoma or 293T cells

Abstract Ras proteins are small GTPases that regulate growth, differentiation, survival, and cell death. Prenylation, the addition of either a farnesyl (15 carbon chain lipid) or geranylgeranyl (20 carbon chain lipid) group, allows Ras and other small GTPases to anchor into the plasma membrane, a necessary step for Ras to activate multiple signaling pathways, including the MAPK cascades. The specific CAAX motif at the C terminus of a protein determines whether it can be farnesylated, geranylgeranylated, or both. There are four highly homologous Ras proteins: K-Ras A, K-Ras B, N-Ras, and H-Ras, each containing a CAAX motif that allows farnesylation. K-Ras B and N-Ras can be alternately geranylgeranylated (GGd) if farnesylation is blocked by a specific small molecule (FTI). Oncogenic Ras mutations are found commonly in cancer, and Ras also can be a major mediator of signals initiated by activated receptor tyrosine kinases (RTKs). Since RTK signaling plays a major role in the biology of sarcomas, we were interested in the mechanisms by which FTIs might affect osteosarcoma cell growth and survival. Osteosarcoma cell lines have variable responses to FTI treatment, including reduced proliferation and programmed cell death at a range of concentrations from 1 nM to 100 nM (Hall et al. 2010). Further, 293T fibroblast cells also have decreased cell viability and increased subG1 and G2/M cell cycle arrest with FTI treatment. 293T cells respond similarly to OS187 our most sensitive osteosarcoma cell line. To assess Ras activity in response to FTI, we co-precipitated activated Ras with the Ras target protein Raf, then probed by western for Ras. Surprisingly, Ras activity was increased in response to FTI in both osteosarcoma cell lines and 293T cells. We also found that the activity of downstream effectors, ERK and p38 MAPK, also increased with FTI (Hall et al, 2010). It was unclear whether the changes in cell growth and survival were due to decrease in farnesylated Ras or the increase in geranylgeranylated Ras. To determine if loss of N-Ras or K-Ras B activity could account for reduced cell yield following FTI, we knocked down each of these proteins in our most responsive osteosarcoma cell line, OS187, and in 293T cells. There was no significant difference in cell yield or cell cycle when either of these proteins was knocked down. To determine if alternate prenylation of N or K-Ras B are responsible for decreased cell yield and/or cell cycle arrest, mutant forms of these proteins will need to be transduced into OS187 and 293T cells for further analysis. In conclusion, FTI caused growth inhibition and cell death for both osteosarcoma cells and 293T cells. FTIs increased Ras activation and the activity of downstream effectors. Since knocking out N or K-Ras B did not reproduce any of these effects, the effects of FTI are not due to the loss of farnesylated Ras. These observations suggest that the effects may be due to increased geranylgeranylated N Ras or K-Ras B. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3856. doi:10.1158/1538-7445.AM2011-3856

Harmful algal bloom species and phosphate-processing effluent: Field and laboratory studies

In 2002, the Florida Department of Environmental Protection began discharging phosphate-processing effluent into Bishop Harbor, an estuary within Tampa Bay. Because of concerns that the effluent would serve as a nutrient source for blooms of the toxic dinoflagellate Karenia brevis, a field monitoring program was established and laboratory bioassays were conducted. Several harmful algal bloom (HAB) species, including Prorocentrum minimum and Heterosigma akashiwo, were observed in bloom concentrations adjacent to the effluent discharge site. Blooms of diatoms were widespread throughout Bishop Harbor. K. brevis was observed with cell concentrations decreasing with increasing proximity to the effluent discharge site. Bioassays using effluent as a nutrient source for K. brevis resulted in decreased cell yields, increased growth rates, and increased time to log-phase growth. The responses of HAB species within Bishop Harbor and of K. brevis to effluent in bioassays suggested that HAB species differ in their response to phosphate-processing effluent.

Fermentation of Glycerol to Succinate by Metabolically Engineered Strains of Escherichia coli

The fermentative metabolism of Escherichia coli was reengineered to efficiently convert glycerol to succinate under anaerobic conditions without the use of foreign genes. Formate and ethanol were the dominant fermentation products from glycerol in wild-type Escherichia coli ATCC 8739, followed by succinate and acetate. Inactivation of pyruvate formate-lyase (pflB) in the wild-type strain eliminated the production of formate and ethanol and reduced the production of acetate. However, this deletion slowed growth and decreased cell yields due to either insufficient energy production or insufficient levels of electron acceptors. Reversing the direction of the gluconeogenic phosphoenolpyruvate carboxykinase reaction offered an approach to solve both problems, conserving energy as an additional ATP and increasing the pool of electron acceptors (fumarate and malate). Recruiting this enzyme through a promoter mutation (pck*) to increase expression also increased the rate of growth, cell yield, and succinate production. Presumably, the high NADH/NAD(+) ratio served to establish the direction of carbon flow. Additional mutations were also beneficial. Glycerol dehydrogenase and the phosphotransferase-dependent dihydroxyacetone kinase are regarded as the primary route for glycerol metabolism under anaerobic conditions. However, this is not true for succinate production by engineered strains. Deletion of the ptsI gene or any other gene essential for the phosphotranferase system was found to increase succinate yield. Deletion of pflB in this background provided a further increase in the succinate yield. Together, these three core mutations (pck*, ptsI, and pflB) effectively redirected carbon flow from glycerol to succinate at 80% of the maximum theoretical yield during anaerobic fermentation in mineral salts medium.

Development and Application of a PCR-Targeted Gene Disruption Method for Studying CelR Function in Thermobifida fusca

Thermobifida fusca is a high-G+C-content, thermophilic, Gram-positive soil actinobacterium with high cellulolytic activity. In T. fusca, CelR is thought to act as the primary regulator of cellulase gene expression by binding to a 14-bp inverted repeat [5'-(T)GGGAGCGCTCCC(A)] that is upstream of many known cellulase genes. Previously, the ability to study the roles and regulation of cellulase genes in T. fusca has been limited largely by a lack of established genetic engineering methods for T. fusca. In this study, we developed an efficient procedure for creating precise chromosomal gene disruptions and demonstrated this procedure by generating a celR deletion strain. The celR deletion strain was then characterized using measurements for growth behavior, cellulase activity, and gene expression. The celR deletion strain of T. fusca exhibited a severely crippled growth phenotype with a prolonged lag phase and decreased cell yields for growth on both glucose and cellobiose. While the maximum endoglucanase activity and cellulase activity were not significantly changed, the endoglucanase activity and cellulase activity per cell were highly elevated. Measurements of mRNA transcript levels in both the celR deletion strain and the wild-type strain indicated that the CelR protein potentially acts as a repressor for some genes and as an activator for other genes. Overall, we established and demonstrated a method for manipulating chromosomal DNA in T. fusca that can be used to study the cellulolytic capabilities of this organism. Components of this method may be useful in developing genetic engineering methods for other currently intractable organisms.

Estrogenic microenvironment influences immunity (39.41)

Abstract Estrogens affect the immune system and immune-mediated/autoimmune diseases. We have found that in vivo estriol (E3) alters dendritic cell (DC) phenotype and function that impact CD4+ T cells. This study evaluates the in vitro effects of E3 (and 17-beta estradiol; E2) on immune function. Spleen (SPL) and lymph node (LN) cells from C57BL/6 mice were CFSE stained and cultured for 3-5 days with media, 100 uM E3 or 100 uM E2 and stimulated with media, anti-CD3 or conA. Decreased proliferation was seen in the E3 (and E2) treated cells as demonstrated by increased mean fluorescence intensity (MFI), fewer daughter cell populations and decreased cell yield in both anti-CD3 and conA stimulated groups. Decreased cell number was not due to cell death as determined by trypan blue and 7-AAD staining. E3 and E2 cultures produced less IL-10 and IL-12p40 by ELISA and phenotypic evaluation of CD11c+ and CD11b+ cells by flow after 48 hours of in vitro E3 stimulation demonstrated only slight effects of E3 on CD80, CD86, PD-L1 and PD-L2. Interestingly, E2 treated cells showed increased CD80 and/or CD86 expression in CD11c+ and CD11b+ cells in the LN and SPL. These studies demonstrate that estrogens can have anti-proliferative effects on SPL and LN populations and influence cytokine and costimulatory molecule expression on antigen presenting cells. Further studies are necessary to compare the in vitro and in vivo effects of estrogens such as E3.

BAG-1 is up-regulated in colorectal tumour progression and promotes colorectal tumour cell survival through increased NF-κB activity

Although expression of the anti-apoptotic protein Bcl-2-associated athanogene-1 (BAG-1) has been reported as up-regulated in a number of malignancies, we show for the first time that BAG-1 is over-expressed in medium/large-sized colorectal adenomas and carcinomas compared with normal epithelium. To investigate whether expression of BAG-1 is important for colorectal tumour cell survival, microarray analysis was carried out on the HCT116 colorectal carcinoma cell line following transfection with BAG-1 small interfering RNA (siRNA). Analysis identified altered expression of a subset of potential nuclear factor-kappaB (NF-kappaB)-regulated genes. Furthermore, knock down of BAG-1 was shown to inhibit NF-kappaB transcriptional activity. Inhibition of NF-kappaB activity using BAG-1 siRNA or the NF-kappaB inhibitor BAY-117082 suppressed HCT116 cell yield and induced apoptosis; combined treatment had no additive effect, suggesting that the decrease in cell yield associated with knock down of BAG-1 expression is mediated via inhibition of NF-kappaB. Of clinical relevance, BAG-1 siRNA sensitized colorectal carcinoma cells to apoptosis induced by potential therapeutic agent TRAIL as well as tumour necrosis factor-alpha, both inducers of NF-kappaB activity. In summary, knock down of BAG-1 leads to inhibition of NF-kappaB, identifying BAG-1 as a novel regulator of NF-kappaB. It is proposed that, by inhibiting NF-kappaB, suppression of BAG-1 could represent a novel strategy to impede colorectal cancer cell survival and as an adjuvant increase sensitivity to current therapeutic regimes.

Fitness Cost of Staphylococcal Cassette Chromosome mec in Methicillin-Resistant Staphylococcus aureus by Way of Continuous Culture

We examined the effect of introducing type I or IV staphylococcal cassette chromosome mec (SCCmec) elements on the growth yield of Staphylococcus aureus in glucose-limited continuous culture. Type I showed increased glucose consumption and ATP demand per gram of cells synthesized and decreased cell yield compared to those of the parent strain. In contrast, type IV SCCmec elements had no adverse energetic effect.

Susceptibility of Escherichia coli to C2-C18 fatty acids.

The antimicrobial activity of C2-C18 fatty acids was determined in vitro in cultures of two strains of Escherichia coli grown on glucose. Antimicrobial activity was expressed as IC50 (a concentration at which only 50% of the initial glucose in the cultures was utilized). Utilization of glucose was inhibited by caprylic acid (IC50 0.30-0.85 g/L) and capric acid (IC50 1.25-2.03 g/L). Neither short-chain fatty acids (C2-C6) nor fatty acids with longer chain (C12-C18) influenced substrate utilization. Caproic acid, however, decreased cell yield in cultures of E. coli in a dose-dependent manner. No inhibition of glucose utilization was produced with unsaturated fatty acids, oleic and linoleic. Calcium ions added in excess reversed the antimicrobial effect of capric acid, but not that of caprylic acid. Antimicrobial activity of caprylic and capric acid decreased when the bacteria were grown in the presence of straw particles, or repeatedly subcultured in a medium containing these compounds at low concentrations. Counts of viable bacteria determined by plating decreased after incubation with caprylic and capric acid (30 min; 1 g/L) at pH 5.2 from > 10(9) to approximately 10(2)/mL. A reduction of a mere 0.94-1.96 log10 CFU was observed at pH 6.5-6.6. It can be concluded that caprylic acid, and to a lesser extent also capric acid, has a significant antimicrobial activity toward E. coli. Effects of other fatty acids were not significant or absent.

Physiological responses of Bacillus stearothermophilus continuous culture to carbon source concentration and temperature shifts

Chemostat cultures of Bacillus stearothermophilus cells using starch as carbon source achieved maximum cell productivity at a dilution rate of 1.0 h −1 with 0.75 g dry weight l −1 h −1. Amylase specific activity ranged from 112 nkat mg −1 to nearly nil values, decreasing when changing dilution rate from 0.1 to 1.5 h −1 and leading to a starch concentration increase from 3 to 9 g l −1. The specific substrate consumption rate increased tenfold (1.04–11.2 g g −1 h −1) with the dilution rate within the range 0.1–1.5 h −1. Starch pulses at low dilution rate (0.2 h −1) produced an increase in specific growth rate (0.26 h −1). Conversely, at high dilution rate (1.0 h −1), there was a marked decrease in the specific growth rate (0.3 h −1). At both dilution rates (0.2 and 1.0 h −1), the steady-state cultures showed a decrease in cell yield and an increase in specific substrate consumption rate. Nevertheless, slow-growing cells (0.2 h −1) showed a double specific substrate consumption rate with half of the cell yield as compared with fast-growing cells (1.0 h −1). Both fast- and slow-growing cells showed a decrease in dry weight during temperature shifts. Reducing sugar concentration remained constant whereas a slight increase in starch concentration and a decrease in amylase specific activity was observed. The enzyme recovered the initial values, once the temperature of 60 °C was restored. However, slow-growing cells (0.2 h −1) showed higher levels of amylase specific activity and, as a result, lower starch concentration than fast-growing cells (1.0 h −1).

Influence of pre-, intra- and post-operative parameters of donor liver on the outcome of isolated human hepatocytes.

The aim of the present study was to analyse, retrospectively on a large panel of patients (149), the influence of the donor liver characteristics on the outcome of human hepatocyte isolation obtained from resected liver biopsies from surgical waste after hepatectomy. Among the pre-operative parameters, the type of disease, age and sex of the patient, previous chemotherapy, alcohol or tobacco consumption did not affect the yield, viability, attachment rate and function of the isolated human hepatocytes. Pre-operative biological and anatomopathological data indicated that, while mild steatosis (</=10% steatotic hepatocytes) did also not affect the outcome of hepatocyte isolation, stronger steatosis (>10% steatotic hepatocytes) tended to decrease hepatocyte yield. Cholestasis, as assessed by gamma-glutamyl transferase serum values, significantly negatively correlated with the percentage of digested liver and the yield of viable cells. Intra-operative clamping time, that is, warm ischaemia, longer than 30 min was found to decrease both the percentage of digested liver and cell yield. Among the post-operative parameters, the percentage of digested liver decreased when biopsy weights were higher than 100 g, the use of glue tended to increase both the percentage of digested tissue and the yield of viable cells.In conclusion, human diseased livers appear to be a valuable source of isolated functional human hepatocytes. We recommend, for an optimal isolation, to use liver biopsies weighing less than 100 g, to glue the section surfaces of the biopsies and to avoid the use of moderate steatotic livers (>10% steatotic hepatocytes) and cholestatic livers, as well as livers undergoing warm ischaemia or clamping during resection due to the decrease in cell yield.

Effect of lipopolysaccharide and cytokines on oxidative metabolism in neonatal rat hepatocytes

Background/Purpose: Lipopolysaccharide (LPS) and cytokines produced during neonatal sepsis trigger free radical production, which eventually results in inhibition of liver metabolism. Studies in adults have indicated a hypermetabolic response to sepsis; however, evidence for a hypermetabolic response in neonates is equivocal. This study was carried out to determine whether LPS and cytokines can cause liver hypermetabolism in neonates. Methods: The initial bacterial insult and cytokine cascade were mimicked by the addition of lipopolysaccharide (Escherichia coli 055:B5), tumour necrosis factor (TNF-α), and interleukin-6 (IL6) during the isolation of hepatocytes by collagenase digestion from 11- to 13-day-old Wistar rats. Hepatocyte oxygen consumption was measured polarographically with cells respiring on palmitate (0.5 mmol/L). Myxothiazol, a specific inhibitor of mitochondrial respiration, was used to distinguish extra- and intramitochondrial oxygen consumption. Morphologic changes were assessed by electron microscopy. Results: The addition of LPS, TNF-α and IL6 during hepatocyte isolation resulted in a 10% decrease in cell yield (P <.05) compared with untreated controls; however, cell viability was unchanged (n = 31). Both total and extramitochondrial oxygen consumption were significantly greater in treated cells compared with untreated controls (P <.05, Student's t test). Electron microscopy indicated that LPS, TNF-α, and IL6 did not cause ultrastructural changes to hepatocytes. Conclusions: The increase in oxygen consumption was predominantly extramitochondrial and likely to be caused by increased oxygen requirement for cytosolic detoxification and repair purposes. This study shows that liver hypermetabolism metabolism can occur in response to LPS and cytokines. However, during in vivo neonatal sepsis, additional free radical damage may blunt this hypermetabolic response. J Pediatr Surg 36:338-340. Copyright © 2001 by W.B. Saunders Company.