Cell type-dependent uptake, localization, and cytotoxicity of 1.9 nm gold nanoparticles

Background: This follow-up study aims to determine the physical parameters which govern the differential radiosensitization capacity of two tumor cell lines and one immortalized normal cell line to 1.9 nm gold nanoparticles. In addition to comparing the uptake potential, localization, and cytotoxicity of 1.9 nm gold nanoparticles, the current study also draws on comparisons between nanoparticle size and total nanoparticle uptake based on previously published data.

Methods: We quantified gold nanoparticle uptake using atomic emission spectroscopy and imaged intracellular localization by transmission electron microscopy. Cell growth delay and clonogenic assays were used to determine cytotoxicity and radiosensitization potential, respectively. Mechanistic data were obtained by Western blot, flow cytometry, and assays for reactive oxygen species.

Results: Gold nanoparticle uptake was preferentially observed in tumor cells, resulting in an increased expression of cleaved caspase proteins and an accumulation of cells in sub G1 phase. Despite this, gold nanoparticle cytotoxicity remained low, with immortalized normal cells exhibiting an LD50 concentration approximately 14 times higher than tumor cells. The surviving fraction for gold nanoparticle-treated cells at 3 Gy compared with that of untreated control cells indicated a strong dependence on cell type in respect to radiosensitization potential.

Conclusion: Gold nanoparticles were most avidly endocytosed and localized within cytoplasmic vesicles during the first 6 hours of exposure. The lack of significant cytotoxicity in the absence of radiation, and the generation of gold nanoparticle-induced reactive oxygen species provide a potential mechanism for previously reported radiosensitization at megavoltage energies.

Design of composite asymmetric cellular beams and beams with large web openings

The design of composite asymmetric cellular beams is not fully covered by existing guidance but is an area of important practical application. Asymmetry in the shape of the cross-section of cellular beams causes development of additional bending moments in the web-posts between closely placed openings. Furthermore, the development of local composite action influences the distribution of forces in the web-flange Tees. The design method presented in this paper takes account of high degrees of asymmetry in the cross-section and also the influence of elongated or rectangular openings.

The cellular level of O-antigen polymerase Wzy determines chain length regulation by WzzB and WzzpHS-2 in Shigella flexneri 2a

The lipopolysaccharide O antigen of Shigella flexneri 2a has two preferred chain lengths, a short (S-OAg) composed of an average of 17 repeated units and a very long (VL-OAg) of about 90 repeated units. These chain length distributions are controlled by the chromosomally encoded WzzB and the plasmid-encoded Wzz(pHS-2) proteins, respectively. In this study, genes wzzB, wzz(pHS-2) and wzy (encoding the O-antigen polymerase) were cloned under the control of arabinose- and rhamnose-inducible promoters to investigate the effect of varying their relative expression levels on O antigen polysaccharide chain length distribution. Controlled expression of the chain length regulators wzzB and wzz(pHS-2) revealed a dose-dependent production of each modal length. Increase in one mode resulted in a parallel decrease in the other, indicating that chain length regulators compete to control the degree of O antigen polymerization. Also, when expression of the wzy gene is low, S-OAg but not VL-OAg is produced. Production of VL-OAg requires high induction levels of wzy. Thus, the level of expression of wzy is critical in determining O antigen modal distribution. Western blot analyses of membrane proteins showed comparable high levels of the WzzB and Wzz(pHS-2) proteins, but very low levels of Wzy. In vivo cross-linking experiments and immunoprecipitation of membrane proteins did not detect any direct interaction between Wzy and WzzB, suggesting the possibility that these two proteins may not interact physically but rather by other means such as via translocated O antigen precursors.

The GalF protein of Escherichia coli is not a UDP-glucose pyrophosphorylase but interacts with the GalU protein possibly to regulate cellular levels of UDP-glucose

We report the functional characterization of the galF gene of strain VW187 (Escherichia coli O7:K1), which encodes a polypeptide displaying structural features common to bacterial UDP-glucose pyrophosphorylases, including the E. coli GalU protein. These enzymes catalyse a reversible reaction converting UTP and glucose-1-phosphate into UDP-glucose and PPi. We show that, although the GalF protein is expressed in vivo, GalF-expressing plasmids cannot complement the phenotype of a galU mutant and extracts from this mutant which only produces GalF are enzymatically inactive. In contrast, the presence of GalU and GalF proteins in the same cell-free extract caused a significant reduction in the rate of pyrophosphorolysis (conversion of UDP-glucose into glucose-1-phosphate) but no significant effect on the kinetics of synthesis of UDP-glucose. The presence of GalF also increased the thermal stability of the enzyme in vitro. The effect of GalF in the biochemical properties of the UDP-glucose pyrophosphorylase required the co-synthesis of GalF and GalU, suggesting that they could interact as components of the oligomeric enzyme. The physical interaction of GalU and GalF was demonstrated in vivo by the co-expression of both proteins as fusion products using a yeast two-hybrid system. Furthermore, using a pair of galF-/galU+ and galF/galU+ isogenic strains, we demonstrated that the presence of GalF is associated with an increased concentration of intracellular UDP-glucose as well as with an enhancement of the thermal stability of the UDP-glucose pyrophosphorylase in vivo. We propose that GalF is a non-catalytic subunit of the UDP-glucose pyrophosphorylase modulating the enzyme activity to increase the formation of UDP-glucose, and this function is important for bacterial adaptation to conditions of stress.

Flanking and internal regions of chromosomal genes mediating aerobactin iron uptake systems in enteroinvasive Escherichia coli and Shigella flexneri

We have investigated the presence of the aerobactin system and the location of the aerobactin genes in enteroinvasive strains of Escherichia coli. Also, we cloned the aerobactin region and its flanking sequences from the chromosome of a strain of Shigella flexneri and compared the molecular organization of the aerobactin genes in the two genera. Of the 11 enteroinvasive E. coli strains studied, 5 possessed the aerobactin genes, which were located on the chromosome in each case. These strains produced and utilized aerobactin and also were susceptible to the bacteriocin cloacin-DF13. Restriction endonuclease mapping and hybridization experiments showed that the regions corresponding to the aerobactin-specific sequences were very similar in both enteroinvasive E. coli and S. flexneri. However, differences were found in the region corresponding to the aerobactin receptor gene. The regions flanking the aerobactin system in enteroinvasive E. coli and S. flexneri exhibited some similarities but were different from those in pColV-K30. Under iron-limiting conditions, aerobactin-producing enteroinvasive E. coli and S. flexneri synthesized outer-membrane proteins of 76 and 77 kDa, respectively, which cross-reacted immunologically with rabbit antiserum raised against the 74 kDa pColV-K30-encoded ferric aerobactin receptor.

Cellular senescence induced by aberrant MAD2 levels impacts on paclitaxel responsiveness in vitro

BACKGROUND: The mitotic arrest deficiency protein 2 (MAD2) is a key component of the mitotic spindle assembly checkpoint, monitoring accurate chromosomal alignment at the metaphase plate before mitosis. MAD2 also has a function in cellular senescence and in a cell’s response to microtubule inhibitory (MI) chemotherapy exemplified by paclitaxel.
METHODS: Using an siRNA approach, the impact of MAD2 down-regulation on cellular senescence and paclitaxel responsiveness was investigated. The endpoints of senescence, cell viability, migration, cytokine expression, cell cycle analysis and anaphase bridge scoring were carried out using standard approaches.
RESULTS: We show that MAD2 down-regulation induces premature senescence in the MCF7 breast epithelial cancer cell line. These MAD2-depleted (MAD2k) cells are also significantly replicative incompetent but retain viability. Moreover, they show significantly higher levels of anaphase bridges and polyploidy compared to controls. In addition, these cells secrete higher levels of IL-6 and IL-8
representing key components of the senescence-associated secretory phenotype (SASP) with the ability to impact on neighbouring cells. In support of this, MAD2kcells show enhanced migratory ability. At 72 h after paclitaxel, MAD2kcells show a significant further induction of senescence compared with paclitaxel naive controls. In addition, there are significantly more viable cells in the MAD2k MCF7 cell line after paclitaxel reflecting the observed increase in senescence.
CONCLUSION: Considering that paclitaxel targets actively dividing cells, these senescent cells will evade cytotoxic kill. In conclusion, compromised MAD2 levels induce a population of senescent cells resistant to paclitaxel.

Examining acute and chronic effects of short- and long-chain fatty acids on peptide YY (PYY) gene expression, cellular storage and secretion in STC-1 cells

PURPOSE: Peptide YY (PYY) is a gastrointestinal hormone with physiological actions regulating appetite and energy homoeostasis. The cellular mechanisms by which nutrients stimulate PYY secretion from intestinal enteroendocrine cells are still being elucidated.

METHODS: This study comprehensively evaluated the suitability of intestinal STC-1 cells as an in vitro model of PYY secretion. PYY concentrations (both intracellular and in culture media) with other intestinal peptides (CCK, GLP-1 and GIP) demonstrated that PYY is a prominent product of STC-1 cells. Furthermore, acute and chronic PYY responses to 15 short (SCFAs)- and long-chain (LCFAs) dietary fatty acids were measured alongside parameters for DNA synthesis, cell viability and cytotoxicity.

RESULTS: We found STC-1 cells to be reliable secretors of PYY constitutively releasing PYY into cell culture media (but not into non-stimulatory buffer). We demonstrate for the first time that STC-1 cells produce PYY mRNA transcripts; that STC-1 cells produce specific time- and concentration-dependent PYY secretory responses to valeric acid; that linoleic acid and conjugated linoleic acid 9,11 (CLA 9,11) are potent PYY secretagogues; and that chronic exposure of SCFAs and LCFAs can be detrimental to STC-1 cells.

CONCLUSIONS: Our studies demonstrate the potential usefulness of STC-1 cells as an in vitro model for investigating nutrient-stimulated PYY secretion in an acute setting. Furthermore, our discovery that CLA directly stimulates L-cells to secrete PYY indicates another possible mechanism contributing to the observed effects of dietary CLA on weight loss.

Elevated soluble cellular adhesion molecules are associated with increased mortality in a prospective cohort of renal transplant recipients

Increased plasma levels of cellular adhesion molecules (CAMs) have been shown to be predictors of all cause mortality in individuals with chronic renal failure 12 and patients with end-stage renal disease receiving haemodialysis 3. In renal transplant recipients the predictive value of CAMs has not been well characterised. The aim of this study was to assess the relationship between CAMs and all-cause mortality during prospective follow-up of a renal transplant cohort.

Uptake and translocation of inorganic and methylated arsenic species by plants

The uptake and translocation into shoots of arsenate, methylarsonate (MA), and dimethylarsinate (DMA) by 46 different plant species were studied. The plants (n = 3 per As species) were exposed for 24 h to 1 mg of As per litre under identical conditions. Total arsenic was measured in the roots and the shoots by acid digestion and inductively coupled plasma mass spectrometry from which, besides total As values, root absorption factors and shoot-to-root transfer factors were calculated. As uptake into the root for the different plant species ranged from 1.2 to 95 (mu g of As per g of dry weight) for As-V, from 0.9 to 44 for MA(V) and from 0.8 to 13 for DMA(V), whereas in shoots the As concentration ranged from 0.10 to 17 for As-V, 0.1 to 13 for MA(V), and 0.2 to 17 for DMA(V). The mean root absorption factor for As-V (1.2 to 95%) was five times higher than for DMA(V) (0.8 to 13%) and 2.5 times higher than for MA(V) (0.9 to 44%). Although the uptake of arsenic in the form of As-V was significantly higher than that of MA(V) and DMA(V), the translocation of the methylated species was more efficient in most plant species studied. Thus, an exposure of plants to DMA(V) or MA(V) can result in higher arsenic concentrations in the shoots than when exposed to As-V. Shoot-to-root transfer factors (TFs) for all plants varied with plant and arsenic species. While As-V had a median TF of 0.09, the TF of DMA(V) was nearly a factor of 10 higher (0.81). The median TF for MA(V) was in between (0.30). Although the TF for MA(V) correlates well with the TF for DMA(V), the plants can be separated into two groups according to their TF of DMA(V) in relation to their TF of As-V. One group can immobilise DMA(V) in the roots, while the other group translocates DMA(V) very efficiently into the shoot. The reason for this is as yet unknown.

Reproductive biomass in Holcus lanatus clones that differ in their phosphate uptake kinetics and mycorrhizal colonization

In normal populations of the common grass Holcus lanatus there is a polymorphism for arsenate resistance, manifested as suppressed phosphate uptake (SPU), and controlled by a major gene with dominant expression. A natural population of SPU plants had greater arbuscular-mycorrhizal colonization than wild type, nonSPU plants. It was hypothesized that, in order to survive alongside plants with a normal rate of phosphate (P) uptake, SPU plants would be more dependent on mycorrhizal associations. We performed an experiment using plants with SPU phenotypes from both arsenate mine spoils and uncontaminated soils, as well as plants with a nonSPU phenotype. They were grown with and without a mycorrhizal inoculum and added N, which altered plant P requirements. We showed that grasses with SPU phenotypes accumulated more shoot P than nonSPU plants, the opposite of the expected result. SPY plants also produced considerably more flower panicles, and had greater shoot and root biomass. The persistence of SPU phenotypes in normal populations is not necessarily related to mycorrhizal colonization as there were no differences in percentage AM colonization between the phenotypes. Being mycorrhizal reduced flower biomass production, as mycorrhizal SPU plants had lower shoot P concentrations and produced fewer flower panicles than non-mycorrhizal, nonSPU plants. We now hypothesize that the SPU phenotype is brought about by a genotype that results in increased accumulation of P in shoots, and that suppression of the rate of uptake is a consequence of this high shoot P concentration, operating by means of a homeostatic feedback mechanism. We also postulate that increased flower production is linked to a high shoot P concentration. SPU plants thus allocate more resources into seed production, leading to a higher frequency of SPU genes. Increased reproductive allocation reduces vegetative allocation and may affect competitive ability and hence survival, explaining the maintenance of the polymorphism. As mycorrhizal SPU plants behave more like nonSPU plants, AM colonization itself could play a major part in the maintenance of the SPU polymorphism.

Organic Matter-Solid Phase Interactions Are Critical for Predicting Arsenic Release and Plant Uptake in Bangladesh Paddy Soils

Agroecological zones within Bangladesh with low levels of arsenic in groundwater and soils produce rice that is high in arsenic with respect to other producing regions of the globe. Little is known about arsenic cycling in these soils and the labile fractions relevant for plant uptake when flooded. Soil porewater dynamics of field soils (n = 39) were recreated under standardized laboratory conditions to investigate the mobility and interplay of arsenic, Fe, Si, C, and other elements, in relation to rice grain element composition, using the dynamic sampling technique diffusive gradients in thin films (DGT). Based on a simple model using only labile DGT measured arsenic and dissolved organic carbon (DOC), concentrations of arsenic in Aman (Monsoon season) rice grain were predicted reliably. DOC was the strongest determinant of arsenic solid-solution phase partitioning, while arsenic release to the soil porewater was shown to be decoupled from that of Fe. This study demonstrates the dual importance of organic matter (OM), in terms of enhancing arsenic release from soils, while reducing bioavailability by sequestering arsenic in solution.