S6K1 is acetylated at lysine 516 in response to growth factor stimulation

The 70kDa ribosomal protein S6 kinase 1 (S6K1) plays important roles in the regulation of protein synthesis, cell growth and metabolism. S6K1 is activated by the phosphorylation of multiple serine and threonine residues in response to stimulation by a variety of growth factors and cytokines. In addition to phosphorylation, we have recently shown that S6K1 is also targeted by lysine acetylation. Here, using tandem mass spectrometry we have mapped acetylation of S6K1 to lysine 516, a site close to the C-terminus of the kinase that is highly conserved amongst vertebrate S6K1 orthologues. Using acetyl-specific K516 antibodies, we show that acetylation of endogenous S6K1 at this site is potently induced upon growth factor stimulation. Although S6K1 acetylation and phosphorylation are both induced by growth factor stimulation, these events appear to be functionally independent. Indeed, experiments using inhibitors of S6K1 activation and exposure of cells to various stresses indicate that S6K1 acetylation can occur in the absence of phosphorylation and vice versa. We propose that K516 acetylation may serve to modulate important kinase-independent functions of S6K1 in response to growth factor signalling.

Effects of cottonseed cake-based diets supplemented with blood meal, alone or with lysine, on the growth of pigs

An experiment was carried out to establish the effect on the growth of pigs of including blood meal or lysine in diets containing gossypol from cottenseed cake. Forty Landrace x Large White pigs (20 of each sex) were randomly allocated to 5 treatments of 8 pigs each in a 2x2 factorial design with two levels of lysine or two levels of blood meal in the diets plus a control diet. The pigs were fed different diets and slaughtered at 75.0+/-2.0 kg live weight for carcase analysis. Supplementing the diets with blood meal resulted in higher live weight gains (p<0.001) and improved feed conversion ratios (p<0.001) than supplementing with lysine. Pigs fed the higher level of cottonseed cake showed a significant (p<0.001) depression in live weight gain and feed conversion ratio compared to those fed a low level of the cake. There was no significant difference (p>0.05) in intake in the pigs fed diets with cottonseed cake including blood meal or synthetic lysine. The kidney and liver weights of the pigs fed the diets with a higher level of cottonseed cake were significantly greater (p<0.001) than in those fed the lower level, but when the diets containing cottonseed cake were supplemented with blood meal or lysine at the same level there was no significant difference (p>0.05) in the weights of these organs. Lysine or other factors derived from blood meal appear to be more efficient than synthetic lysine in reducing the adverse effects of gossypol.

Histone H4 gene expression and morphological changes on shoot apices of strawberry (Fragaria x ananassa Duch.) during floral induction

To investigate flower induction in June-bearing strawberry plants, morphological changes in shoot apices and Historic H4 expression in the central zone during flower initiation were observed. Strawberry plants were placed under flower inducible, short-day conditions (23 degrees C/17 degrees C, 10 h day length) for differing number of days (8, 16, 20, 24 or 32 days) and then these plants were transferred to non-inducible, long-day conditions (25 degrees C/20 degrees C, 14 h day length). The shoot apices of plants placed under short-day conditions for 8 days were flat, similar to shoot apices of plants in the vegetative phase of development, and Histone H4 was not expressed in the central zone during the experimental period. On the other hand, the shoot apices of plants placed under short-day conditions for 16 days remained flat, similar to shoot apices of plants placed under short-day conditions for 8 days, but Histone H4 was expressed in the central zone at the end of the short-day treatment. Morphological changes in the shoot apices of these plants were observed 8 days after the change in day-length. These plants developed differentiated flower organs after they were grown for another 30 days under long-day conditions. These results indicate that changes in the expression pattern of the Histone H4 gene occur before morphological changes during flower induction and that the expression of the gene in the central zone can be used as one of the indicators of the flowering process in strawberries. (c) 2006 Elsevier B.V. All rights reserved.

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.

Spontaneous condensation in DNA-polystyrene-b-poly(l-lysine) polyelectrolyte block copolymer mixtures

We investigated the condensation of calf thymus DNA by amphiphilic polystyrene(m)-b-poly(l-lysine)(n) block copolymers (PSm-b- PLys(n), m, n = degree of polymerization), using small-angle X-ray scattering, polarized optical microscopy and laser scanning confocal microscopy. Microscopy studies showed that the DNA condenses in the form of fibrillar precipitates, with an irregular structure, due to electrostatic interactions between PLys and DNA. This is not modified by the presence of hydrophobic PS block. Scattering experiments show that the structure of the polyplexes corresponds to a local order of DNA rods which becomes more compact upon increasing n. It can be concluded that for DNA/ PSm-b- PLys(n) polyplexes, the balance between the PLys block length and the excess charge in the system plays an essential role in the formation of a liquid crystalline phase.

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes, Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.

Nonspherical assemblies generated from polystyrene-b-poly(L-lysine) polyelectrolyte block copolymers

This report describes the aqueous solution self-assembly of a series of polystyrene(m)-b-poly(L-lysine)n block copolymers (m = 8-10; n = 10-70). The polymers are prepared by ring-opening polymerization of epsilon-benzyloxycarbonyl-L-lysine N-carboxyanhydride using amine terminated polystyrene macroinitiators, followed by removal of the benzyloxycarbonyl side chain protecting groups. The critical micelle concentration of the block copolymers determined using the pyrene probe technique shows a parabolic dependence on peptide block length exhibiting a maximum at n = approximately 20 (m = 8) or n = approximately 60 (m = 10). The shape and size of the aggregates has been studied by dynamic and static light scattering, small-angle neutron scattering (SANS), and analytical ultracentrifugation (AUC). Surprisingly, Holtzer and Kratky analysis of the static light scattering results indicates the presence of nonspherical, presumably cylindrical objects independent of the poly(L-lysine)n block length. This is supported by SANS data, which can be fitted well by assuming cylindrical scattering objects. AUC analysis allows the molecular weight of the aggregates to be estimated as several million g/mol, corresponding to aggregation numbers of several 10s to 100s. These aggregation numbers agree with those that can be estimated from the length and diameter of the cylinders obtained from the scattering results.

The adsorption geometry and chemical state of lysine on Cu{110}

Chemisorbed layers of lysine adsorbed on Cu{110} have been studied using X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. XPS indicates that the majority (70%) of the molecules in the saturated layer at room temperature (coverage 0.27 ML) are in their zwitterionic state with no preferential molecular orientation. After annealing to 420 K a less densely packed layer is formed (0.14 ML), which shows a strong angular dependence in the characteristic π-resonance of oxygen K edge NEXAFS and no indication of zwitterions in XPS. These experimental results are best compatible with molecules bound to the substrate through the oxygen atoms of the (deprotonated) carboxylate group and the two amino groups involving Cu atoms in three different close packed rows. This μ4 bonding arrangement with an additional bond through the !-amino group is different from geometries previously suggested for lysine on Cu{110}.

The impact of catechol-O-methyltransferase genotype on the acute responsiveness of vascular reactivity to a green tea extract

The beneficial effects of green tea catechins, such as the proposed improvement in endothelial function, may be influenced by phase II metabolism during and after absorption. The methylation enzyme, catechol-O-methyltransferase (COMT), has a missense mutation rs4680 (G to A), proposed to result in a 40 % reduction in enzyme activity. In the present pilot study, twenty subjects (ten of each homozygous COMT genotype) were recruited. Green tea extract capsules (836 mg green tea catechins) were given in a fasted state, and a high-carbohydrate breakfast was given after 60 min. Blood samples and vascular function measurements were taken at regular intervals. The change in digital volume pulse stiffness index (SI) from baseline was shown to be different between genotype groups at 120 and 240 min, with a lower SI in the GG individuals (P ≤ 0·044). The change in blood pressure from baseline also differed between genotype groups, with a greater increase in systolic (P = 0·023) and diastolic (P = 0·034) blood pressure at 120 min in the GG group. The AA group was shown to have a greater increase in insulin concentrations at 120 min (P = 0·019) and 180 min (P = 0·008) compared with baseline, despite similar glucose profiles. No genotypic differences were found in vascular reactivity measured using laser Doppler iontophoresis, total nitrite, lipids, plasma total antioxidant capacity or inflammatory markers after ingestion of the green tea extract. In conclusion, SI and insulin response to the glucose load differed between the COMT genotype groups, and this may be suggestive of a green tea extract and genotype interaction.

A preliminary investigation of the impact of catechol-O-methyltransferase genotype on the absorption and metabolism of green tea catechins

Purpose Green tea is thought to possess many beneficial effects on human health. However, the extent of green tea polyphenol biotransformation may affect its proposed therapeutic effects. Catechol-O-methyltransferase (COMT), the enzyme responsible for polyphenolic methylation, has a common polymorphism in the genetic code at position 158 reported to result in a 40% reduction in enzyme activity in in vitro studies. The current preliminary study was designed to investigate the impact of COMT genotype on green tea catechin absorption and metabolism in humans. Methods Twenty participants (10 of each homozygous COMT genotype) were recruited, and plasma concentration profiles were produced for epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), epicatechin (EC) and 4′-O-methyl EGCG after 1.1 g of Sunphenon decaffeinated green tea extract (836 mg green tea catechins), with a meal given after 60 min. Results For the entire group, EGCG, EGC, EC, ECG and 4′-O-methyl EGCG reached maximum concentrations of 1.09, 0.41, 0.33, 0.16 and 0.08 μM at 81.5, 98.5, 99.0, 85.5 and 96.5 min, respectively. Bimodal curves were observed for the non-gallated green tea catechins EGC and EC as opposed to single-peaked curves for the gallated green tea catechins EGCG and ECG. No significant parametric differences between COMT genotype groups were found. Conclusions In conclusion, the COMT Val(158/108)Met does not appear to have a dramatic influence on EGCG absorption and elimination. However, further pharmacokinetic research is needed to substantiate these findings.

The impact of the catechol-O-methyltransferase genotype on vascular function and blood pressure after acute green tea ingestion

SCOPE: Evidence for the benefits of green tea catechins on vascular function is inconsistent, with genotype potentially contributing to the heterogeneity in response. Here, the impact of the catechol-O-methyltransferase (COMT) genotype on vascular function and blood pressure (BP) after green tea extract ingestion are reported. METHODS AND RESULTS: Fifty subjects (n = 25 of the proposed low-activity [AA] and of the high-activity [GG] COMT rs4680 genotype), completed a randomized, double-blind, crossover study. Peripheral arterial tonometry, digital volume pulse (DVP), and BP were assessed at baseline and 90 min after 1.06 g of green tea extract or placebo. A 5.5 h and subsequent 18.5 h urine collection was performed to assess green tea catechin excretion. A genotype × treatment interaction was observed for DVP reflection index (p = 0.014), with green tea extract in the AA COMT group attenuating the increase observed with placebo. A tendency for a greater increase in diastolic BP was evident at 90 min after the green tea extract compared to placebo (p = 0.07). A genotypic effect was observed for urinary methylated epigallocatechin during the first 5.5 h, with the GG COMT group demonstrating a greater concentration (p = 0.049). CONCLUSION: Differences in small vessel tone according to COMT genotype were evident after acute green tea extract.

An epigenetic signature of developmental potential in neural stem cells and early neurons.

A cardinal property of neural stem cells (NSCs) is their ability to adopt multiple fates upon differentiation. The epigenome is widely seen as a read-out of cellular potential and a manifestation of this can be seen in embryonic stem cells (ESCs), where promoters of many lineage-specific regulators are marked by a bivalent epigenetic signature comprising trimethylation of both lysine 4 and lysine 27 of histone H3 (H3K4me3 and H3K27me3, respectively). Bivalency has subsequently emerged as a powerful epigenetic indicator of stem cell potential. Here, we have interrogated the epigenome during differentiation of ESC-derived NSCs to immature GABAergic interneurons. We show that developmental transitions are accompanied by loss of bivalency at many promoters in line with their increasing developmental restriction from pluripotent ESC through multipotent NSC to committed GABAergic interneuron. At the NSC stage, the promoters of genes encoding many transcriptional regulators required for differentiation of multiple neuronal subtypes and neural crest appear to be bivalent, consistent with the broad developmental potential of NSCs. Upon differentiation to GABAergic neurons, all non-GABAergic promoters resolve to H3K27me3 monovalency, whereas GABAergic promoters resolve to H3K4me3 monovalency or retain bivalency. Importantly, many of these epigenetic changes occur before any corresponding changes in gene expression. Intriguingly, another group of gene promoters gain bivalency as NSCs differentiate toward neurons, the majority of which are associated with functions connected with maturation and establishment and maintenance of connectivity. These data show that bivalency provides a dynamic epigenetic signature of developmental potential in both NSCs and in early neurons. Stem Cells 2013;31:1868-1880.

Diallyl disulfide-induced apoptosis in a breast-cancer cell line (MCF-7) may be caused by inhibition of histone de-acetylation

The health benefits of garlic have been proven by epidemiological and experimental studies. Diallyl disulphide (DADS), the major organosulfur compound found in garlic oil, is known to lower the incidence of breast cancer both in vitro and in vivo. The studies reported here demonstrate that DADS induces apoptosis in the MCF-7 breast-cancer cell line through interfering with cell-cycle growth phases in a way that increases the sub-G0 population and substantially halts DNA synthesis. DADS also induces phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane and activates caspase-3. Further studies revealed that DADS modulates the cellular levels of Bax, Bcl-2, Bcl-xL and Bcl-w in a dose-dependent manner, suggesting the involvement of Bcl-2 family proteins in DADS induced apoptosis. Histone deacetylation inhibitors (HDACi) are known to suppress cancer growth and induce apoptosis in cancer cells. Here it is shown that DADS has HDACi properties in MCF-7 cells as it lowers the removal of an acetyl group from an acetylated substrate and induces histone-4 (H4) hyper-acetylation. The data thus indicate that the HDACi properties of DADS may be responsible for the induction of apoptosis in breast cancer cells.