Differential induction of 12-O-tetradecanoylphorbol 13-acetate sequence gene expression in murine melanocytes and melanoma cells

We previously showed that growth of the nontumorigenic, immortal murine melanocyte line Mel-ab correlates with the depletion of protein kinase C (PKC), whereas quiescence is associated with elevated levels of this enzyme (Brooks G, et al., Cancer Res 51: 3281–3288, 1991). Here we report responses that occur in these cells downstream of PKC activation or downregulation. We examined induction of 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible sequence (TIS) gene expression in Mel-ab melanocytes and in their transformed counterparts, B16 melanoma cells. Exposure of quiescent Mel-ab cells to the PKC-activating phorbol esters TPA or sapintoxin A at 81 nM for 2 h increased levels of mRNA for six of seven TIS genes examined (twofold to 80-fold increase in steady-state RNA levels for TIS 1, 7, 8, 11, 21, and 28 (c-fos); TIS 10 expression was not affected). No induction of 115 gene expression was observed either in growing Mel-ab cells maintained in 324 nM phorbol 12,13-dibutyrate or in B16 cells previously unexposed to phorbol esters, in which normal PKC levels were endogenously depressed. The cAMP-elevating agents choleratoxin (10 nM) and dibutyryl cyclic AMP (2.5 mM) increased levels of TIS mRNA (with the exception of TIS 10) in both proliferating Mel-ab and B16 cells, suggesting that downregulation of the PKC pathway is specific and not a consequence of a general inhibition of all signalling pathways.

Protein kinase C down-regulation, and not transient activation, correlates with melanocyte growth

The nontumorigenic, immortal line of murine melanocytes, Mel-ab, requires the continual presence of biologically active phorbol esters for growth (R. E. Wilson et al., Cancer Res., 49: 711–716, 1989). Comparable treatments of B16 murine melanoma cells result in partial inhibition of cell proliferation. The role of protein kinase C (PKC) in the modulation of growth of cells from these two melanocytic cell lines has been investigated. Significant levels of PKC were present in quiescent Mel-ab cells as determined by Western blotting, whereas no immunoreactive protein was detected in cell extracts from either proliferating Mel-ab or B16.F1 cells. Phosphorylation of a Mr 80,000 protein, which by one- and two-dimensional gel analysis comigrated with the known Mr 80,000 protein substrate of PKC in fibroblasts, was induced in 12-O-tetradecanoylphorbol-13-acetate-stimulated quiescent Mel-ab cells but not in proliferating Mel-ab cells or B16.F1 melanoma cells. Direct measurement of PKC activity in these cells demonstrated a 10-fold greater level of activity in quiescent Mel-ab cells (262 ± 50 pmol/min/mg SD) compared with growing cells (22.8 ± 11.8 pmol/min/mg SD). An intermediate level of activity was detected in proliferating B16.F1 melanoma cells (148.5 ± 20.4 pmol/min/mg SD). The subcellular distribution of PKC was dependent upon the growth state of the cells such that quiescent Mel-ab cells displayed a higher level of activity in the cytosol, whereas growing Melab cells displayed greater activity in the particulate fraction. Like many other transformed lines, B16.F1 melanoma cells constitutively expressed the majority of enzyme activity in the particulate fraction. Measurement of [3H]phorbol ester binding in intact cells paralleled the PKC activation data such that quiescent Mel-ab cells displayed binding of 1612 ± 147 cpm/106 cells, whereas proliferating Mel-ab and B16.F1 melanoma cells displayed binding of 652 ± 28 and 947 ± 81 cpm/106 cells, respectively. Membrane-permeant diacylglycerol analogues, which activated but did not down-regulate PKC, were devoid of growth-stimulating effects on melanocytes, even in the presence of the specific diacylglycerol kinase inhibitor, R59022. Together, these data show that PKC down-regulation, and not activation, correlates with the growth of melanocytes in culture.

Inhibition of 125I-epidermal growth factor binding to murine fibroblasts and keratinocytes by irradiation with ultraviolet-B light

Treatment of murine Swiss 3T3 fibroblasts and XB/2 keratinocytes with UV-B light (302 nm) resulted in a dose-dependent inhibition of [125I] epidermal growth factor (EGF) binding. The light dose required to achieve 50% inhibition of binding in both cell types was 80–85 J/m2 Decreased [125I] platelet-derived growth factor binding was not evoked even by light doses of up to 280 J/m2 UV-B irradiation did not stimultate phosphorylation of the 80 kd protein substrate for protein kinase C. Furthermore, its effect on [125I]EGF binding was not altered as a consequence of protein kinase C down-regulation following prolonged exposure of cells to phorbol esters. These results indicate that UV-B-induced transmodulation of the epidermal growth factor receptor is a specific event mediated through a protein kinase C-indepen dent pathway. Transfer of culture medium from irradiated cells to untreated control cells showed this effect was not induced as a result of transforming growth factor α release and subsequent binding to the EGF receptor in these cells.

Daphnane diterpenes of Thymelaea hirsuta

Five 12-hydroxy-daphnane esters were isolated from the leaves and twigs of Egyptian Thymelaea hirsuta. These compounds were identified as gnidicin, gniditrin, genkwadaphnin, the aliphatic C-12 ester, 12-O-heptadecenoyl-5-hydroxy-6,7-epoxy-resiniferonol-9,13,14-orthobenzoate and the novel aliphatic C-12 ester 12-O-butenyl-5-hydroxy-6,7-epoxy-resiniferonol-9,13-14-orthobenzoate.

An ingenane diterpene from Belizian Mabea excelsa

The new ingenane diterpene, 5-deoxy-13-hydroxyingenol, was isolated from the alcohol preserved fresh latex of the stems of Mabea excelsa and characterized from its semi-synthetic triacetate. This is the first instance of an ingenane diterpene obtained from species other than those of Euphorbia and Elaeophorbia. This diterpene occurred in the latex in the form of an inseparable mixture of six aliphatic mono-esters of the tertiary C-13 hydroxy group.

Asymmetric phase-transfer-catalyzed synthesis of five-membered cyclic gamma-Amino acid precursors

The first example of an intramolecular enantioselective Michael addition of nitronates onto conjugated systems utilizing a chiral phase-transfer catalyst is described. A range of five-membered gamma-nitro esters with up to three stereocentres have been prepared and the relative and absolute configurations proven by chemical and crystallographic methods. The products are rapidly obtained and are precursors to five-membered cyclic gamma-amino acids.

Free amino acids and sugars in rye grain: implications for acrylamide formation

Acrylamide forms from free asparagine and sugars during cooking, and products derived from the grain of cereals, including rye, contribute a large proportion of total dietary intake. In this study, free amino acid and sugar concentrations were measured in the grain of a range of rye varieties grown at locations in Hungary, France, Poland, and the United Kingdom and harvested in 2005, 2006, and 2007. Genetic and environmental (location and harvest year) effects on the levels of acrylamide precursors were assessed. The data showed free asparagine concentration to be the main determinant of acrylamide formation in heated rye flour, as it is in wheat. However, in contrast to wheat, sugar, particularly sucrose, concentration also correlated both with asparagine concentration and with acrylamide formed. Free asparagine concentration was shown to be under genetic (G), environmental (E), and integrated (G × E) control. The same was true for glucose, whereas maltose and fructose were affected mainly by environmental factors and sucrose was largely under genetic control. The ratio of variation due to varieties (genotype) to the total variation (a measure of heritability) for free asparagine concentration in the grain was 23%. Free asparagine concentration was closely associated with bran yield, whereas sugar concentration was associated with low Hagberg falling number. Rye grain was found to contain much higher concentrations of free proline than wheat grain, and less acrylamide formed per unit of asparagine in rye than in wheat flour.

Asymmetric synthesis of cyclic β-amino acids and cyclic amines via sequential diastereoselective conjugate addition and ring closing metathesis

Diastereoselective conjugate addition of lithium (S)-N-allyl-N-alpha-methylbenzylamide to a range of alpha,beta-unsaturated esters followed by ring closing metathesis is used to afford efficiently a range of substituted cyclic beta-amino esters in high d.e. Alternatively, conjugate addition to alpha,beta-unsaturated Weinreb amides, functional group conversion and ring closing metathesis affords cyclic amines in high d.e. The further application of this methodology to the synthesis of a range of carbocyclic beta-amino esters via conjugate addition, enolate alkylation and ring closing metathesis is also described. Application of this methodology affords, after deprotection, (S)-homoproline, (S)-homopipecolic acid, (S)-coniine and (1S,2S)-trans-pentacin.

Bacterial, SCFA and gas profiles of a range of food ingredients following in vitro fermentation by human colonic microbiota.

It is now apparent that there is a strong link between health and nutrition and this can be seen clearly when we talk of obesity. The food industry is trying to capitalise on this by adapting high sugar/fat foods to become healthier alternatives. In confectionery food ingredients can be used for a range of purposes including sucrose replacement. Many of these ingredients may also evade digestion in the upper gut and be fermented by the gut microbiota upon entering the colon. This study was designed to screen a range of ingredients and their activities on the gut microbiota. In this study we screened a range of these ingredients in triplicate batch culture fermentations with known prebiotics as controls. Changes in bacteriology were monitored using FISH. SCFA were measured by GC and gas production was assessed during anaerobic batch fermentations. Bacterial enumeration showed significant increases (P ≤ 0.05) in bifidobacteria and lactobacilli with polydextrose and most polyols with no significant increases in Clostridium histolyticum/perfringens. SCFA and gas formation indicated that the substrates added to the fermenters were being utilised by the gut microbiota. It therefore appears these ingredients exert some prebiotic activity in vitro. Further studies, particularly in human volunteers, are necessary.

Environmental factors influencing the inactivation of Cronobacter sakazakii by high hydrostatic pressure

The effect of High Hydrostatic Pressure (HHP) on the survival of Cronobacter sakazakii was investigated. Deviations from linearity were found on the survival curves and the Mafart equation accurately described the kinetics of inactivation. Comparisons between strains and treatments were made based on the time needed for a 5-log10 reduction in viable count. The ability of C. sakazakii to tolerate high pressure was straindependent with a 26-fold difference in resistance among four strains tested. Pressure resistance was greatest in the stationary growth phase and at the highest growth temperatures tested (30 and 37 °C). Cells treated in neutral pH buffer were 5-fold more resistant than those treated at pH 4.0, and 8-fold more sensitive than those treated in buffer with sucrose added (aw=0.98). Pressure resistance data obtained in buffer at the appropriate pH adequately estimated the resistance of C. sakazakii in chicken and vegetables soups. In contrast, a significant protective effect against high pressure was conferred by rehydrated powdered milk. As expected, treatment efficacy improved as pressure increased. z values of 112, 136 and 156 MPa were obtained for pH 4.0, pH 7.0 and aw=0.98 buffers, respectively. Cells with sublethal injury to their outer and cytoplasmic membranes were detected after HHP under all the conditions tested. The lower resistance of C. sakazakii cells when treated in media of pH 4.0 seemed to be due to a decreased barostability of the bacterial envelopes. Conversely, the higher resistance displayed in media of reduced water activity may relate to a higher stability of bacterial envelopes.

Sugars in crop plants

We review current knowledge of the most abundant sugars, sucrose, maltose, glucose and fructose, in the world's major crop plants. The sucrose-accumulating crops, sugar beet and sugar cane, are included, but the main focus of the review is potato and the major cereal crops. The production of sucrose in photosynthesis and the inter-relationships of sucrose, glucose, fructose and other metabolites in primary carbon metabolism are described, as well as the synthesis of starch, fructan and cell wall polysaccharides and the breakdown of starch to produce maltose. The importance of sugars as hormone-like signalling molecules is discussed, including the role of another sugar, trehalose, and the trehalose biosynthetic pathway. The Maillard reaction, which occurs between reducing sugars and amino acids during thermal processing, is described because of its importance for colour and flavour in cooked foods. This reaction also leads to the formation of potentially harmful compounds, such as acrylamide, and is attracting increasing attention as food producers and regulators seek to reduce the levels of acrylamide in cooked food. Genetic and environmental factors affecting sugar concentrations are described.

Effect of dextransucrase cellobiose acceptor products on the growth of human gut bacteria

The selective fermentation by human gut bacteria of gluco-oligosaccharides obtained from the reaction between the glucosyl group of sucrose and cellobiose, catalyzed by dextransucrases (DSR) from Leuconostoc mesenteroides, has been evaluated. Oligosaccharides were fractionated according to their molecular weight, and their effect on the growth of different bacterial groups was studied. To determine the structure (position and configuration of glycosidic linkages)�function relationship, their properties were compared to those of DSR maltose acceptor products (DSRMal) and of recognized prebiotic carbohydrates (fructo-oligosaccharides, FOS). Cellobiose acceptor products (DSRCel) showed bifidogenic properties similar to those of FOS. However, no significant differences related to molecular weight or isomeric configurations were found for DSRCel and DSRMal products.

Measurement of paraben concentrations in human breast tissue at serial locations across the breast from axilla to sternum

The concentrations of five esters of p-hydroxybenzoic acid (parabens) were measured using HPLC-MS/MS at four serial locations across the human breast from axilla to sternum using human breast tissue collected from 40 mastectomies for primary breast cancer in England between 2005 and 2008. One or more paraben esters were quantifiable in 158/160 (99%) of the tissue samples and in 96/160 (60%) all five esters were measured. Variation was notable with respect to individual paraben esters, location within one breast and similar locations in different breasts. Overall median values in nanograms per gram tissue for the 160 tissue samples were highest for n-propylparaben [16.8 (range 0–2052.7)] and methylparaben [16.6 (range 0–5102.9)]; levels were lower for n-butylparaben [5.8 (range 0–95.4)], ethylparaben [3.4 (range 0–499.7)] and isobutylparaben 2.1 (range 0–802.9). The overall median value for total paraben was 85.5 ng g−1 tissue (range 0–5134.5). The source of the paraben cannot be identified, but paraben was measured in the 7/40 patients who reported never having used underarm cosmetics in their lifetime. No correlations were found between paraben concentrations and age of patient (37–91 years), length of breast feeding (0–23 months), tumour location or tumour oestrogen receptor content. In view of the disproportionate incidence of breast cancer in the upper outer quadrant, paraben concentrations were compared across the four regions of the breast: n-propylparaben was found at significantly higher levels in the axilla than mid (P = 0.004 Wilcoxon matched pairs) or medial (P = 0.021 Wilcoxon matched pairs) regions (P = 0.010 Friedman ANOVA).

Cinnamate ester containing liquid crystalline side chain polymers

The synthesis of methacrylate esters of 4-cyanophenyl-(4-(ω-hydroxyalkyloxy)) cinnamates, with spacer lengths of 2 and 6 methylene units and the synthesis of the corresponding acrylate ester with a spacer of 2 methylene units are described. The methacrylate monomers were polymerized by free radical polymerization, both as homopolymers and as copolymers with the analogous benzoate monomer of spacer length 6. The acrylate ester could not be polymerized successfully under the same reaction conditions. Polymers were characterized by NMR spectroscopy, gel permeation chromatography, differential scanning calorimetry, and thermo-optic observations. Of the monomers prepared, only the cinnamate with a hexamethylene spacer shows a mesophase, seen on supercooling of the melt. All of the polymers prepared were liquid crystalline, with smectic behavior predominating in the polymethacrylates with the longer spacer group. A narrow nematic region is seen just below the clearing temperature with a range of 3–9°C, nematic character is increased in the copolymer series with the degree of incorporation of the cinnamate monomer with the spacer group of length 2.

Oxidation of low density lipoprotein by iron at lysosomal pH: implications for atherosclerosis

Low density lipoprotein (LDL) has recently been shown to be oxidised by iron within the lysosomes of macrophages and this is a novel potential mechanism for LDL oxidation in atherosclerosis. Our aim was to characterise the chemical and physical changes induced in LDL by iron at lysosomal pH and to investigate the effects of iron chelators and α-tocopherol on this process. LDL was oxidised by iron at pH 4.5 and 37°C and its oxidation monitored by spectrophotometry and HPLC. LDL was oxidised effectively by FeSO4 (5-50 µM) and became highly aggregated at pH 4.5, but not at pH 7.4. Cholesteryl esters decreased and after a pronounced lag 7-ketocholesterol increased greatly. Total hydroperoxides (measured by tri-iodide assay) increased up to 24 h and then decreased only slowly. The lipid composition after 12 h at pH 4.5 and 37°C was similar to that of LDL oxidised by copper at pH 7.4 and 4°C, i.e. rich in hydroperoxides but low in oxysterols. Previously oxidised LDL aggregated rapidly and spontaneously at pH 4.5, but not at pH 7.4. Ferrous was much more effective than ferric iron at oxidising LDL when added after the oxidation was already underway. The iron chelators diethylenetriaminepentaacetic acid and, to a lesser extent, desferrioxamine inhibited LDL oxidation when added during its initial stages, but were unable to prevent LDL aggregating after it had been partially oxidised. Surprisingly, desferrioxamine increased the rate of LDL modification when added late in the oxidation process. α-Tocopherol enrichment of LDL initially increased the oxidation of LDL, but inhibited it later. The presence of oxidised and highly aggregated lipid within lysosomes has the potential to perturb the function of these organelles and to promote atherosclerosis.