Unsupplemented Cells Research Articles

Magnesium Modulates Bacillus subtilis Cell Division Frequency.

By chance, we discovered a window of extracellular magnesium (Mg2+) availability that modulates the division frequency of Bacillus subtilis without affecting its growth rate. In this window, cells grown with excess Mg2+ produce shorter cells than do those grown in unsupplemented medium. The Mg2+-responsive adjustment in cell length occurs in both rich and minimal media as well as in domesticated and undomesticated strains. Of other divalent cations tested, manganese (Mn2+) and zinc (Zn2+) also resulted in cell shortening, but this occurred only at concentrations that affected growth. Cell length decreased proportionally with increasing Mg2+ from 0.2 mM to 4.0 mM, with little or no detectable change being observed in labile, intracellular Mg2+, based on a riboswitch reporter. Cells grown in excess Mg2+ had fewer nucleoids and possessed more FtsZ-rings per unit cell length, consistent with the increased division frequency. Remarkably, when shifting cells from unsupplemented to supplemented medium, more than half of the cell length decrease occurred in the first 10 min, consistent with rapid division onset. Relative to unsupplemented cells, cells growing at steady-state with excess Mg2+ showed an enhanced expression of a large number of SigB-regulated genes and the activation of the Fur, MntR, and Zur regulons. Thus, by manipulating the availability of one nutrient, we were able to uncouple the growth rate from the division frequency and identify transcriptional changes that suggest that cell division is accompanied by the general stress response and an enhanced demand to sequester and/or increase the uptake of iron, Mn2+, and Zn2+. IMPORTANCE The signals that cells use to trigger cell division are unknown. Although division is often considered intrinsic to the cell cycle, microorganisms can continue to grow and repeat rounds of DNA replication without dividing, indicating that cycles of division can be skipped. Here, we show that by manipulating a single nutrient, namely, Mg2+, cell division can be uncoupled from the growth rate. This finding can be applied to investigate the nature of the cell division signal(s).

Effect of nutrient supplements on molasses fermentation by salt preconditioned cells of Saccharomyces cerevisiae

Molasses, the major by-product of sugarcane processing industry, is an efficient and economical source for ethanol production. In the present work, the effect of yeast extract, tween-80 and ergosterol on molasses fermentation was studied using salt preconditioned Saccharomyces cerevisiae MTCC 11815 cells. Design expert 10.0 software under RSM plan was used to optimize the nutrient concentrations and four responses viz. residual brix, ethanol, glycerol and trehalose produced were recorded. Thus, with optimized nutrient concentrations, the salt preconditioned cells showed significantly better ethanol production (11.45%, v/v) as compared to controls viz. preconditioned un-supplemented cells (9.92%, v/v) and unconditioned cells (8.13%, v/v). Further, the molasses fermentation was validated at 10L scale and 11.67% (v/v) of ethanol was produced with 86.83% fermentation efficiency with 0.523% (w/v), 0.251% (v/v) and 3.8mg/100ml of yeast extract, tween-80 and ergosterol supplementation, respectively.

Polyunsaturated Fatty Acids of Both the Omega-3 and the Omega-6 Family Abrogate the Cytokine-Induced Upregulation of miR-29a-3p by Endothelial Cells.

Cellular processes fundamentally depend on protein expression control. At this, protein expression is regulated on the transcriptional and the post-transcriptional level. PUFAs are already known to affect gene transcription. The present study was conducted to answer the question whether PUFAs are also able to impact on the miRNA-mediated post-transcriptional fine-tuning of mRNA copy numbers. To this end, cellular miRNA profiles were screened by means of next-generation sequencing and NanoString analysis to compare PUFA-enriched to unsupplemented endothelial cells exposed to an inflammatory milieu. Validation took place by droplet digital PCR, allowing for an absolute quantification of RNA copy numbers. The analyses revealed that the stimulation-induced upregulation of miR-29a-3p is blocked by PUFA enrichment of endothelial cells. What is more, mRNA copy numbers of miR-29a-3p targets, namely the coagulation factors PAI-1, TF, and vWF, as well as the proinflammatory cytokines IL-1β, IL-6, and IL-8, were reduced in PUFA-enriched endothelial cells compared to unsupplemented cells, counteracting the stimulatory effect of an inflammatory environment. These data hint toward a new mechanism of action by which PUFAs modulate the functionality of endothelial cells. Apparently, the inflammation-modulating properties of PUFAs are also mediated at the post-transcriptional level.

Strategies for the hypothermic preservation of cell sheets of human adipose stem cells.

Cell Sheet (CS) Engineering is a regenerative medicine strategy proposed for the treatment of injured or diseased organs and tissues. In fact, several clinical trials are underway using CS-based methodologies. However, the clinical application of such cell-based methodologies poses several challenges related with the preservation of CS structure and function from the fabrication site to the bedside. Pausing cells at hypothermic temperatures has been suggested as a valuable method for short-term cell preservation. In this study, we tested the efficiency of two preservation strategies, one using culture medium supplementation with Rokepie and the other using the preservation solution Hypothermosol, in preserving human adipose stromal/stem cells (hASC) CS-like confluent cultures at 4°C, during 3 and 7 days. Both preservation strategies demonstrated excellent ability to preserve cell function during the first 3 days in hypothermia, as demonstrated by metabolic activity results and assessment of extracellular matrix integrity and differentiation potential. At the end of the 7th day of hypothermic incubation, the decrease in cell metabolic activity was more evident for all conditions. Nonetheless, hASC incubated with Rokepie and Hypothermosol retained a higher metabolic activity and extracellular matrix integrity in comparison with unsupplemented cells. Differentiation results for the later time point showed that supplementation with both Rokepie and Hypothermosol rescued adipogenic differentiation potential but only Rokepie was able to preserve hASC osteogenic potential.

The pyrimidine biosynthetic pathway and its regulation in Pseudomonas jessenii.

The control of the pyrimidine biosynthetic pathway by pyrimidine bases was examined in Pseudomonas jessenii ATCC 700870. The pyrimidine biosynthetic enzymes aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate (OMP) decarboxylase activities were found to be higher in the succinate-grown ATCC 700870 cells than the glucose-grown cells. All the enzyme activities were depressed in uracil-supplemented ATCC 700870 glucose-grown cells relative to the unsupplemented cells which was indicative of possible repression of enzyme synthesis by uracil. In the succinate-grown, ATCC 700870 cells, transcarbamoylase, dihydroorotase and dehydrogenase activities were decreased by uracil and orotate supplementation while decarboxylase activity was decreased following uracil addition. A pyrimidine auxotroph was isolated by conventional chemical mutagenesis and resistance to 5-fluoroorotic acid whose pyrimidine requirement was met by uracil or cytosine. The mutant strain was deficient for orotate phosphoribosyltransferase activity. Pyrimidine limitation of the mutant strain cells for 1 or 2h caused about a two-fold increase in aspartate transcarbamoylase or dihydroorotase activity independent of carbon source relative to excess uracil growth conditions. At the level of enzyme activity, aspartate transcarbamoylase activity in P. jessenii ATCC 700870 was inhibited strongly by pyrophosphate, ATP, UTP, GTP and UMP under saturating substrate concentrations.

Pyrimidine nucleotide synthesis in the emerging pathogen Pseudomonas monteilii.

Regulation of pyrimidine biosynthesis by pyrimidines in the emerging, opportunistic human pathogen Pseudomonas monteilii ATCC 700476 was evident. When wild-type cells were grown on succinate in the presence of uracil or orotic acid, the activities of all 5 pyrimidine biosynthetic enzymes were depressed while the activities of 3 of the enzymes decreased in glucose-grown cells supplemented with uracil or orotic acid compared with unsupplemented cells. Pyrimidine limitation of succinate- or glucose-grown pyrimidine auxotrophic cells lacking orotate phosphoribosyltransferase activity resulted in more than a doubling of the pyrimidine biosynthetic enzyme activities relative to their activities in uracil-grown cells. Independent of carbon source, pyrimidine-limited cells of the pyrimidine auxotrophic cells deficient for dihydroorotase activity generally resulted in a slight elevation or depression of the pyrimidine biosynthetic enzyme activities compared with their activities in cells grown under saturating uracil conditions. Aspartate transcarbamoylase activity in P. monteilii was regulated at the enzyme activity level, since the enzyme was strongly inhibited by CTP, UMP, GMP, GDP, ADP, and UTP. In summary, the regulation of pyrimidine biosynthesis in P. monteilii could be used to control its growth or to differentiate it biochemically from other related species of Pseudomonas.

O46 Selenium supplementation and modified high-intensity interval training (m-HIIT) to modulate systemic oxidative stress in sedentary overweight/obese adults: In vitro and in vivo studies

Obesity is characterised by systemic oxidative stress (OS) resulting from high levels of reactive oxidative species (ROS) and reduced antioxidant capacity. OS plays a fundamental role in cardiovascular disease (CVD) development by initiating atherosclerotic plaque formation. Increased dietary antioxidants’ intake or up-regulation of endogenous antioxidant enzymes may counteract OS and lower CVD risk. Essential micronutrient selenium supplementation and regular physical activity increase endogenous antioxidant enzyme Glutathione Peroxidases (GPx) activity. However, it is unknown whether modified high-intensity interval training (m-HIIT), an emerging time-efficient exercise regime, modulates redox status among sedentary overweight/obese adults, and whether it has adjunctive effects when combined with selenium supplementation. This work investigates the effect of selenium supplementation on antioxidant enzymes gene expression and ROS generation in vitro, and assesses the role of a dietary and exercise intervention in modulating OS and CVD risk in vivo. Monocyte cells were cultured with/out sodium selenite (100 nM) and stimulated with Paraquat/S-Nitroso-N-acetyl-DL-penicillamine (PQ/SNAP, 1 mM/0.7 mM) to induce OS. Treatment with PQ/SNAP significantly increased ROS generation compared to untreated cells, confirming OS induction; whereas selenium supplementation significantly increased GPx1 (146%) and GPx4 gene expression (77%) and reduced ROS production when compared to un-supplemented stressed cells. Preliminary results from the three-armed randomised intervention study, 8 weeks m-HIIT (10 × 1min at 60% peak aerobic power, three days/week) and/or selenium supplementation (100 μg selenomethionine/day), showed that a single m-HIIT bout significantly increased plasma GPx activity 15 min post-exercise and combined interventions reduced OS. Selenium supplementation and m-HIIT may, therefore, be effective in counteracting OS by significantly increasing GPxs expression in an in vitro and in vivo system.

Aerobic transformation of zinc into metal sulfide by photosynthetic microorganisms

Industrial activity over the last two centuries has increased heavy metal contamination worldwide, leading to greater human exposure. Zinc is particularly common in industrial effluents and although an essential nutrient, it is highly toxic at elevated concentrations. Photoautotrophic microbes hold promise for heavy metal bioremediation applications because of their ease of culture and their ability to produce sulfide through metabolic processes that in turn are known to complex with the metal ion, Hg(II). The green alga Chlamydomonas reinhardtii, the red alga Cyanidioschyzon merolae, and the cyanobacterium Synechococcus leopoliensis were all able to synthesize sulfide and form zinc sulfide when exposed to Zn(II). Supplementation of their respective media with sulfite and cysteine had deleterious effects on growth, although ZnS still formed in Cyanidioschyzon cells to the same extent as in unsupplemented cells. The simultaneous addition of sulfate and Zn(II) had similar effects to that of Zn(II) alone in all three species, whereas supplying sulfate prior to exposure to Zn(II) enhanced metal sulfide production. The coupled activities of serine acetyltransferase and O-acetylserine(thiol)lyase (SAT/OASTL) did not increase significantly in response to conditions in which enhanced ZnS formation occurred; sulfate added prior to and simultaneously with Zn(II). However, even low activity could provide sufficient sulfate assimilation over this relatively long-term study. Because the extractable activity of cysteine desulfhydrase was elevated in cells that produced higher amounts of zinc sulfide, cysteine is the probable source of the sulfide in this aerobic process.

Abstract 787: The role of monocytes in promoting natural killer cell activation in response to antibody-coated tumor cells and IL-12

Abstract Introduction: Previous studies from our group has demonstrated that co-stimulation of NK cells via the interleukin-12 receptor (IL-12R) and the FcgRIIIa activates the extracellular signal-regulated kinase (ERK) signaling pathway in vitro, in murine tumor models and in phase I clinical trials. The ERK pathway in turn promotes the secretion of interferon-gamma (IFN-g) thereby mediating potent anti-tumor effects. Hypothesis: NK cell cytokine secretion would be significantly enhanced following simultaneous stimulation of the NK cells via the activating receptor, NKG2D and their ligands (such as MICA/B) expressed on monocytes. Methods: Purified NK cells, Monocytes and peripheral blood mononuclear cells (PBMC) were co-cultured with antibody (Ab)-coated tumors cells in the presence of IL-12. Cell free supernatants were analyzed for the presence of inflammatory cytokines. Results: Co-stimulation of purified NK cells with trastuzumab-coated HER2+ SKBR3 breast cancer cells and IL-12 (10 ng/mL) resulted in synergistic production of IFN-g (>20,000 pg/mL) as compared to the single conditions (<3000 pg/mL). Cytokine production in response to antibody-coated tumor cells and IL-12 was significantly enhanced in the presence of autologous monocytes (2-3-fold, P<0.05) but not T cells or B cells. Maximum cytokine secretion was observed at 48 hours, and preceded by a 6-fold increase in IFN-g transcript. This enhancement of cytokine response was dependent on cell-cell contact as determined by a Transwell assay. NK cells co-cultured with monocytes (1:1=200,000 cells per well) also secreted significantly higher amounts of TNF-a (>2 fold) and MIP1 alpha (>4 fold) as compared to un-supplemented NK cells exposed to Ab and IL-12. A dose-response effect was observed with increasing numbers of added monocytes. Pre-treatment of monocytes with LPS and/or IFN-alpha led to increased expression of NKG2D ligands (MICA and MICB) and increased the ability of monocytes to act as co-stimulators of NK cell cytokine secretion. The stimulatory effects of MICA/B+ monocytes were duplicated by the use of a MICA over-expressing cell line (C1R-MICA) but not the parental MICA-negative cell line (2-3.2 fold increase). Incubation of C1R MICA cells with a MICA neutralizing antibody prior to co-culture with NK cells led to a significant reduction in IFN-g secretion. The stimulatory effects of monocytes were also observed in whole PBMC in that depletion of monocytes from PBMC markedly inhibited the production of IFN-g by the NK cell compartment whereas supplementation of PBMC with additional monocytes led to dose-dependent increases in cytokine production. Conclusions: These data suggest that stimulation of NKG2D by monocyte ligands can enhance the NK cell cytokine response to Ab-coated targets. Enhancement of NK monocyte interactions could increase the efficacy of Ab-based anti-cancer therapies. 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 787. doi:10.1158/1538-7445.AM2011-787

Homology-Directed Repair Is Diminished in PTEN-Positive Breast Tumor Cells.

Abstract BackgroundThe tumor suppressor gene PTEN is mutated or lost in a substantial proportion of human cancers, including basal-like breast cancer (BLBC). Recently, aside from its lipid phosphatase activity, PTEN has been implicated in DNA repair via a regulatory role of RAD51, the central repair protein in homology-directed repair (HDR). As BLBCs are transcriptionally similar to HDR-deficient BRCA1-associated breast cancers, it is hypothesized that loss of PTEN may extend a repair-deficient phenotype to BRCA1 wild-type BLBC. Tumors which exhibit impaired DNA double-strand break (DSB) repair are potential targets for chemotherapy with DNA-damaging agents, and novel strategies such as poly(ADP)ribose polymerase (PARP) inhibition. We therefore set out to quantify the repair capability of an induced DSB by HDR in PTEN-deficient breast tumor cells, and to assess their sensitivity to agents targeting DNA repair.MethodsTo assess HDR of DNA DSBs we used several assays developed to rapidly quantify cellular repair capability – including the I-SceI-induced DSB DR-GFP assay (Pierce et al., 1999) and a newly developed assay measuring GFP-gene targeting to a zinc finger nuclease (ZFN) induced DSB (Brunet et al., PNAS 2009). PTEN-deficient MDA-MB-468 cells stably transfected with tetracycline-inducible PTEN (She et al., 2003) underwent transient co-transfection with an I-SceI expression vector and DR-GFP. Assessment of gene targeting to an induced break in the p84 gene on chromosome 19 was also assessed as an alternative HDR assay by co-transfection of GFP p84 and ZFNp84 constructs. PTEN and RAD51 protein expression in doxycycline supplemented and unsupplemented cells were assessed by Western blot. We performed clonogenic survival assays following PARP inhibitor continuous exposure at concentrations of 10-5 to 10-8 M for 7-14 days.ResultsPTEN induction following doxycycline exposure was confirmed by Western blot. RAD51 expression was similar in PTEN-positive and PTEN-negative cells. Cell cycle analysis of PTEN-induced cells indicated that similar proportions of PTEN-positive and PTEN-negative cells were in S-phase of the cell cycle. PTEN-positive cells demonstrated decreased HDR of an induced DSB relative to PTEN-negative cells using the DR-GFP reporter system with a mean ratio of GFP-positive cells (PTEN positive: PTEN negative) of 0.52 (SD 0.30). The ratio was statistically significant by log-transformed paired t-test (p=0.02, 95% CI 0.08, 0.60). Preliminary experiments demonstrate a two-fold reduction in gene targeting to a ZFN-induced chromosome break in PTEN positive cells as compared to PTEN negative cells. Initial clonogenic assays demonstrate significantly increased sensitivity to PARP inhibition in the PTEN-positive cells.ConclusionIn contrast with the expectation that nuclear PTEN promotes RAD51 expression, direct assessment of HDR DSB repair demonstrates decreased repair proficiency as well as increased sensitivity to PARP inhibition in PTEN-positive as compared to cells with PTEN loss. Further work will focus on HDR assessment in additional PTEN-deficient cell lines to determine whether this is an MDA-MB-468-specific phenomenon. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1120.

Cargo surface hydrophobicity is sufficient to overcome the nuclear pore complex selectivity barrier

To fulfil their function, nuclear pore complexes (NPCs) must discriminate between inert proteins and nuclear transport receptors (NTRs), admitting only the latter. This specific permeation is thought to depend on interactions between hydrophobic patches on NTRs and phenylalanine-glycine (FG) or related repeats that line the NPC. Here, we tested this premise directly by conjugating different hydrophobic amino-acid analogues to the surface of an inert protein and examining its ability to cross NPCs unassisted by NTRs. Conjugation of as few as four hydrophobic moieties was sufficient to enable passage of the protein through NPCs. Transport of the modified protein proceeded with rates comparable to those measured for the innate protein when bound to an NTR and was relatively insensitive both to the nature and density of the amino acids used to confer hydrophobicity. The latter observation suggests a non-specific, small, and plant interaction network between cargo and FG repeats.

Competition between 24:5n-3 and ALA for Δ6 desaturase may limit the accumulation of DHA in HepG2 cell membranes

The use of Delta 6 desaturase (D6D) twice in the conversion of alpha-linolenic acid (ALA; 18:3n-3) to docosahexaenoic acid (DHA; 22:6n-3) suggests that this enzyme may play a key regulatory role in the synthesis and accumulation of DHA from ALA. We examined this using an in vitro model of fatty acid metabolism to measure the accumulation of the long-chain metabolites of ALA in HepG2 cell phospholipids. The accumulation of ALA, eicosapentaenoic acid (20:5n-3), docosapentaenoic acid (22:5n-3), and 24:5n-3 in cell phospholipids was linearly related to the concentration of supplemented ALA over the range tested (1.8-72 microM). The accumulation of the post-D6D products of 22:5n-3, 24:6n-3 and DHA, in cell phospholipids was saturated at concentrations of >18 microM ALA. Supplementation of HepG2 cells with preformed DHA revealed that, although the accumulation of DHA in cell phospholipids approached saturation, the level of DHA in cell phospholipids was significantly greater compared with the accumulation of DHA from ALA, indicating that the accumulation of DHA from ALA was not limited by incorporation. The parallel pattern of accumulation of 24:6n-3 and DHA in response to increasing concentrations of ALA suggests that the competition between 24:5n-3 and ALA for D6D may contribute to the limited accumulation of DHA in cell membranes.

Docosahexaenoic acid (22:6n‐3) enrichment of membrane phospholipids increases gap junction coupling capacity in cultured astrocytes

Although it is agreed that n-3 polyunsaturated fatty acids (PUFAs) are important for brain function, it has yet to be demonstrated how they are involved in precise cellular mechanisms. We investigated the role of enhanced n-3 PUFA in astrocyte membranes on the gap junction capacity of these cells. Astrocytes isolated from newborn rat cortices were grown in medium supplemented with docosahexaenoic acid (DHA), the main n-3 PUFA in cell membranes, or arachidonic acid (AA), the main n-6 PUFA, plus an antioxidant (alpha-tocopherol or N-acetyl-cystein) to prevent peroxidation. The resulting three populations of astrocytes differed markedly in their n-3:n-6 PUFA ratios in phosphatidylethanolamine and phosphatidylcholine, the main phospholipids in membranes. DHA-supplemented cells had a physiological high n-3:n-6 ratio (1.58), unsupplemented cells had a low n-3:n-6 ratio (0.66) and AA-supplemented cells had a very low n-3:n-6 ratio (0.36), with excess n-6 PUFA. DHA-supplemented astrocytes had a greater gap junction capacity than unsupplemented cells or AA-supplemented cells. The enhanced gap junction coupling of DHA-enriched cells was associated with a more functional distribution of connexin 43 at cell interfaces (shown by immunocytochemistry) and more of the main phosphorylated isoform of connexin 43. These findings suggest that the high n-3:n-6 PUFA ratio that occurs naturally in astrocyte membranes is needed for optimal gap junction coupling in these cells.

Growth temperature affects accumulation of exogenous fatty acids and fatty acid composition in Schizosaccharomyces pombe

The incorporation of exogenously supplied fatty acids, palmitic acid, palmitoleic acid, oleic acid and linoleic acid, was examined in the yeast Schizosaccharomyces pombe at two growth temperatures, 20 °C and 30 °C. Fatty acids supplied to S. pombe in the growth medium were found to be preferentially incorporated into the cells, becoming a dominant species. The relative increase in exogenous fatty acids in cells came at the expense of endogenous oleic acid as a proportion of total fatty acids. Lowering the temperature at which the yeast were grown resulted in decreased levels of incorporation of the fatty acids palmitic acid, palmitoleic acid and linoleic acid compared to cells supplemented at 30 °C. In addition, the relative amount of the endogenously produced unsaturated fatty acid oleic acid, while greatly reduced compared to unsupplemented cells, was increased in cells supplemented with fatty acids at 20 °C compared to supplemented cells at 30 °C. The differential production of oleic acid in S. pombe cells indicates that regulation of unsaturated fatty acid levels, possibly by control of the stearoyl-CoA desaturase, is an important control point in membrane composition in response to temperature and diet in this species.

Growth temperature affects accumulation of exogenous fatty acids and fatty acid composition in Schizosaccharomyces pombe

The incorporation of exogenously supplied fatty acids, palmitic acid, palmitoleic acid, oleic acid and linoleic acid, was examined in the yeast Schizosaccharomyces pombe at two growth temperatures, 20 degrees C and 30 degrees C. Fatty acids supplied to S. pombe in the growth medium were found to be preferentially incorporated into the cells, becoming a dominant species. The relative increase in exogenous fatty acids in cells came at the expense of endogenous oleic acid as a proportion of total fatty acids. Lowering the temperature at which the yeast were grown resulted in decreased levels of incorporation of the fatty acids palmitic acid, palmitoleic acid and linoleic acid compared to cells supplemented at 30 degrees C. In addition, the relative amount of the endogenously produced unsaturated fatty acid oleic acid, while greatly reduced compared to unsupplemented cells, was increased in cells supplemented with fatty acids at 20 degrees C compared to supplemented cells at 30 degrees C. The differential production of oleic acid in S. pombe cells indicates that regulation of unsaturated fatty acid levels, possibly by control of the stearoyl-CoA desaturase, is an important control point in membrane composition in response to temperature and diet in this species.

Cloning and expression of human sialic acid pathway genes to generate CMP-sialic acids in insect cells.

The addition of sialic acid residues to glycoproteins can affect important protein properties including biological activity and in vivo circulatory half-life. For sialylation to occur, the donor sugar nucleotide cytidine monophospho-sialic acid (CMP-SA) must be generated and enzymatically transferred to an acceptor oligosaccharide. However, examination of insect cells grown in serum-free medium revealed negligible native levels of the most common sialic acid nucleotide, CMP-N-acetylneuraminic acid (CMP-Neu5Ac). To increase substrate levels, the enzymes of the metabolic pathway for CMP-SA synthesis have been engineered into insect cells using the baculovirus expression system. In this study, a human CMP-sialic acid synthase cDNA was identified and found to encode a protein with 94% identity to the murine homologue. The human CMP-sialic acid synthase (Cmp-Sas) is ubiquitously expressed in human cells from multiple tissues. When expressed in insect cells using the baculovirus vector, the encoded protein is functional and localizes to the nucleus as in mammalian cells. In addition, co-expression of Cmp-Sas with the recently cloned sialic acid phosphate synthase with N-acetylmannosamine feeding yields intracellular CMP-Neu5Ac levels 30 times higher than those observed in unsupplemented CHO cells. The absence of any one of these three components abolishes CMP-Neu5Ac production in vivo. However, when N-acetylmannosamine feeding is omitted, the sugar nucleotide form of deaminated Neu5Ac, CMP-2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (CMP-KDN), is produced instead, indicating that alternative sialic acid glycoforms may eventually be possible in insect cells. The human CMP-SAS enzyme is also capable of CMP-N-glycolylneuraminic acid (CMP-Neu5Gc) synthesis when provided with the proper substrate. Engineering the CMP-SA metabolic pathway may be beneficial in various cell lines in which CMP-Neu5Ac production limits sialylation of glycoproteins or other glycans.

Effect of docosahexaenoic acid on the synthesis of phosphatidylserine in rat brain in microsomes and C6 glioma cells.

Docosahexaenoic acid (22:6n-3) is the major polyunsaturated fatty acid (PUFA) in the CNS and accumulates particularly in phosphatidylserine (PS). We have investigated the effect of the 22:6n-3 compositional status on the synthesis of PS. The fatty acid composition of brain microsomes from offspring of rats artificially reared on an n-3-deficient diet showed a dramatic reduction of 22:6n-3 content (1.7 +/- 0.1%) when compared with control animals (15.0 +/- 0.2%). The decrease was accompanied by an increase in docosapentaenoic acid (22:5n-6) content, which replaced the 22:6n-3 phospholipids with 22:5n-6 molecular species, as demonstrated using HPLC/electrospray mass spectrometry. The n-3 deficiency did not affect the total amount of polyunsaturated phospholipids in brain microsomes; however, it was associated with a decrease in the total polyunsaturated PS content and with increased levels of 1-stearoyl-2-docosapentanoyl (18:0/22:5n-6) species, particularly in phosphatidylcholine. Incorporation of [3H]serine into PS in rat brain microsomes from n-3-deficient animals was slightly but significantly less than that of the control animals. Similarly, C6 glioma cells cultured for 24 h in 22:6n-3-supplemented media (10-40 microM) showed a significant increase in the synthesis of [3H]PS when compared with unsupplemented cells. Our data show that neuronal and glial PS synthesis is sensitive to changes in the docosahexaenoate levels of phospholipids and suggest that 22:6n-3 may be a modulator of PS synthesis.

Influence of altered plasma membrane fatty acid composition on cesium transport characteristics and toxicity in Saccharomyces cerevisiae.

The influence of altered plasma membrane fatty acid composition on cesium uptake and toxicity was investigated in Saccharomyces cerevisiae. Detailed kinetic studies revealed that both the Vmax and Km values for Cs+ transport increased (by approximately twofold in the latter case) when S. cerevisiae was grown in medium supplemented with the polyunsaturated fatty acid linoleate. In addition, Cs+ uptake by linoleate-enriched cells was considerably less sensitive to the competitive effects of other monovalent cations (K+, Rb+, and NH4+) than that by unsupplemented cells. Stimulation of Cs+ uptake in the presence of certain K+ and Rb+ concentrations was only evident in linoleate-enriched S. cerevisiae. At 100 mM CsCl, the initial rate of Cs+ uptake was greater in linoleate-supplemented cells than in unsupplemented cells and this was reflected in a more rapid displacement of cellular K+. However, little difference in net Cs+ accumulation between linoleate-supplemented and unsupplemented cells was evident during prolonged incubation in buffer or during growth. Thus, Cs+ toxicity was similar in linoleate-supplemented and unsupplemented cells. The results were consistent with the Cs+ (K+) transport mechanism adopting an altered conformational state in linoleate-enriched S. cerevisiae.

Manipulation of lipid composition of rat heart myocytes aged in culture and its effect on α1-adrenoceptor stimulation

The fatty acid composition of the phosphoinositides was evaluated in cultured neonatal rat cardiomyocytes during the aging-like process in vitro, comparing data obtained from control and γ-linolenic acid supplemented cardiomyocytes. The response to α 1 stimulation was evaluated in both control and supplemented cells to verify the relationship between the alterations of the phosphoinositide fatty acid composition concomitant to culture aging and the cell response to exogenous stimuli. Arachidonate level decreased as a function of age in all the phosphoinositides, which appeared to be more saturated as cells aged in culture. Inositol phosphate production in response to α 1 stimulation decreased as cells aged in culture. Supplementation of culture medium with γ-linolenic acid caused significant modifications in the fatty acid pattern of the phosphoinositides, which appeared less saturated than the corresponding fractions isolated from unsupplemented cells during the aging-like process. The modifications induced by the supplementation in the phosphoinositide fatty acid composition prevented the age-related reduction of inositol phosphate production upon stimulation. These results clearly indicate a major role for the lipid composition in determining the response to α 1 stimulation, suggesting a nutritional approach to overcome some of the impairments of molecular events related to the process of aging.

Induction of lipid peroxidation during heavy metal stress in Saccharomyces cerevisiae and influence of plasma membrane fatty acid unsaturation.

The degree of plasma membrane fatty acid unsaturation and the copper sensitivity of Saccharomyces cerevisiae are closely correlated. Our objective was to determine whether these effects could be accounted for by differential metal induction of lipid peroxidation. S. cerevisiae S150-2B was enriched with the polyunsaturated fatty acids (PUFAs) linoleate (18:2) and linolenate (18:3) by growth in 18:2- or 18:3-supplemented medium. Potassium efflux and colony count data indicated that sensitivity to both copper (redox active) and cadmium (redox inactive) was increased in 18:2-supplemented cells and particularly in 18:3-supplemented cells. Copper- and cadmium-induced lipid peroxidation was rapid and associated with a decline in plasma membrane lipid order, detected by fluorescence depolarization measurements with the membrane probe trimethylammonium diphenylhexatriene. Levels of thiobarbituric acid-reactive substances (lipid peroxidation products) were up to twofold higher in 18:2-supplemented cells than in unsupplemented cells following metal addition, although this difference was reduced with prolonged incubation up to 3 h. Conjugated-diene levels in metal-exposed cells also increased with both the concentration of copper or cadmium and the degree of cellular fatty acid unsaturation; maximal levels were evident in 18:3-supplemented cells. The results demonstrate heavy metal-induced lipid peroxidation in a microorganism for the first time and indicate that the metal sensitivity of PUFA-enriched S. cerevisiae may be attributable to elevated levels of lipid peroxidation in these cells.