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.

Effects of biologically active tumour-promoting and non-promoting phorbol esters on in vivo growth of melanocytic cells

Sapintoxin A (SAP A), a naturally occurring biologically active but non-promoting phorbol ester, acts as an effective in vitro mitogen for freshly derived human melanocytes. Seven days after addition of 50 nM SAP A there was a four to fivefold increase in melanocyte number over that observed in untreated control cultures comparable to that achieved with a 50 nM concentration of 12-0-tetradecanoylphorbol 13-acetate (TPA). The fluorescent stage 2 promoter sapintoxin D (SAP D) also supported the growth of these cells, with a 50 nM dose producing an increase in cell number comparable to that observed with 200 nM TPA. Similar results were obtained with an established, but non-tumorigenic, line of murine melanocytes. The same compounds exerted a potent anti-proliferative effect against transformed melanocyte lines of murine and human origin associated with morphological alterations and an increase in melanin production consistent with induced cytodifferentiation.

Both tumour-promoting and non-promoting phorbol esters inhibit 125I-EGF binding and stimulate the phosphorylation of a 80kd protein kinase C substrate protein in intact Swiss 3T3 cells

Sapintoxin A (SAP A) and 12-deoxyphorbol 13-phenylacetate (DOPP), are two biologically active but non-turnour-promoting phorbol esters that potently bind to and activate the phorbol ester receptor, protein kinase C (PKC). SAP A and DOPP cause a dose-dependent increase in the phosphorylation of an 80 kd (80K) substrate protein for PKC in Swiss 3T3 cells. A similar dose—response effect was seen with sapintoxin D (SAP D), the stage 2 promoting analogue of 12-O-tetradecanoylphorbol-13-acetate and the complete promoter phorbol 12,13-dibutyrate (PDB). The doses resulting in a half maximal phosphorylation of this protein (Ka were 20 nM (SAP A), 45 nM (DOPP), 23 nM (SAP D) and 37 nM (PDB). Both non-promoting and phorbol esters induced a dose-dependent inhibition of [125I]epidermal growth factor (EGF) binding to its receptor in Swiss 3T3 cells. The doses required for 50% inhibition of binding (Ki) were: 8 nM (SAP A), 16 nM (DOPP), 14 nM (SAP D) and 17 nM (PDB). The results clearly demonstrate that induction of phosphorylation of the Pu 80K phosphoprotein and inhibition of [125I]EGF binding in Swiss 3T3 cells following exposure to phorbol esters is independent of the tumour-promoting activity of these compounds. The fact that SAP A, DOPP, SAP D and PDB are mitogenic for a variety of cell types and that exposure to these compounds leads to 80K phosphorylation and inhibition of [125I]EGF binding, suggests that these early biological events may play a role in the mitogenic response induced by these compounds.

The effects of phorbol esters with different activities on protein kinase C

The sapintoxins are a series of naturally occurring fluorescent phorbol esters with a range of selective biological activities (e.g. pro-inflammatory but non-tumour promoting). Their ability to activate protein kinase C (PKC) in vitro has been studied. Both tumour promoting and non-promoting phorbol derivatives activate the enzyme in vitro at low concentrations. 12-deoxyphorbol-13-phenylacetate-20 acetate (DOPPA) acts as a partial agonist in the activation of protein kinase C. Structurally distinct phorbol esters may therefore preferentially activate different forms of protein kinase C. -sapinine, a biologically inactive compound, binds to protein kinase C without stimulating the enzyme and prevents subsequent activation by phorbol esters such as 12-O-tetradecanoyl phorbol-13-acetate (TPA).

Effects of vitamin E and fish oil inclusion in broiler diets on meat fatty acid composition and on the flavour of a composite sample of breast meat

BACKGROUND: Enriching poultry meat with long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) can increase low population intakes of LC n-3 PUFA, but fishy taints can spoil reheated meat. This experiment determined the effect of different amounts of LC n-3 PUFA and vitamin E in the broiler diet on the fatty acid composition and sensory characteristics of the breast meat. Ross 308 broilers (120) were randomly allocated to one of five treatments from 21 to 42 days of age. Diets contained (g kg−1) 0, 9 or 18 LC n-3 PUFA (0LC, 9LC, 18LC), and 100, 150 or 200 mg LD--tocopherol acetate kg−1 (E). The five diets were 0LC100E, 9LC100E, 18LC100E, 18LC150E, 18LC200E, with four pens per diet, except 18LC100E (eight pens). Breast meat was analysed for fatty acids (uncooked) and sensory analysis by R-index (reheated). RESULTS: LC n-3 PUFA content (mg kg−1 meat) was 514 (0LC100E) and 2236 (9LC and 18LC). Compared with 0LC100E, meat from 18LC100E and 18LC150E tasted significantly different, while 23% of panellists detected fishy taints in 9LC100E and 18LC200E. CONCLUSION: Chicken meat can be enriched with nutritionally meaningful amounts of LC n-3 PUFA, but > 100 mg dl--tocopherol acetate kg−1 broiler diet is needed to protect reheated meat from oxidative deterioration. Copyright © 2010 Society of Chemical Industry

Effect of incremental levels of fish oil in the diet on ruminal lipid metabolism in growing steers

Based on the potential benefits to human health, there is interest in developing sustainable nutritional strategies to enhance the concentration of long-chain n-3 fatty acids in ruminant-derived foods. Four Aberdeen Angus steers fitted with rumen and duodenal cannulae were used in a 4 × 4 Latin square experiment with 21 d experimental periods to examine the potential of fish oil (FO) in the diet to enhance the supply of 20 : 5n-3 and 22 : 6n-3 available for absorption in growing cattle. Treatments consisted of total mixed rations based on maize silage fed at a rate of 85 g DM/kg live weight0·75/d containing 0, 8, 16 and 24 g FO/kg diet DM. Supplements of FO reduced linearly (P < 0·01) DM intake and shifted (P < 0·01) rumen fermentation towards propionate at the expense of acetate and butyrate. FO in the diet enhanced linearly (P < 0·05) the flow of trans-16 : 1, trans-18 : 1, trans-18 : 2, 20 : 5n-3 and 22 : 6n-3, and decreased linearly (P < 0·05) 18 : 0 and 18 : 3n-3 at the duodenum. Increases in the flow of trans-18 : 1 were isomer dependent and were determined primarily by higher amounts of trans-11 reaching the duodenum. In conclusion, FO alters ruminal lipid metabolism of growing cattle in a dose-dependent manner consistent with an inhibition of ruminal biohydrogenation, and enhances the amount of long-chain n-3 fatty acids at the duodenum, but the increases are marginal due to extensive biohydrogenation in the rumen.

The influence of pomegranate by-product and punicalagins on selected groups of human intestinal microbiota.

We have examined the gut bacterial metabolism of pomegranate by-product (POMx) and major pomegranate polyphenols, punicalagins, using pH-controlled, stirred, batch culture fermentation systems reflective of the distal region of the human large intestine. Incubation of POMx or punicalagins with faecal bacteria resulted in formation of the dibenzopyranone-type urolithins. The time course profile confirmed the tetrahydroxylated urolithin D as the first product of microbial transformation, followed by compounds with decreasing number of phenolic hydroxy groups: the trihydroxy analogue urolithin C and dihydroxylated urolithin A. POMx exposure enhanced the growth of total bacteria, Bifidobacterium spp. and Lactobacillus spp., without influencing the Clostridium coccoides–Eubacterium rectale group and the C. histolyticum group. In addition, POMx increased concentrations of short chain fatty acids (SCFA) viz. acetate, propionate and butyrate in the fermentation medium. Punicalagins did not affect the growth of bacteria or production of SCFA. The results suggest that POMx oligomers, composed of gallic acid, ellagic acid and glucose units, may account for the enhanced growth of probiotic bacteria.

Monophosphothreonyl extracellular signal-regulated kinases 1 and 2 (ERK1/2) are formed endogenously in intact cardiac myocytes and are enzymically active

ERK1 and ERK2 (ERK1/2) are central to the regulation of cell division, growth and survival. They are activated by phosphorylation of the Thr- and the Tyr- residues in their Thr-Glu-Tyr activation loops. The dogma is that dually-phosphorylated ERK1/2 constitute the principal activities in intact cells. We previously showed that, in neonatal rat cardiac myocytes, endothelin-1 and phorbol 12-myristate 13-acetate (PMA) powerfully and rapidly (maximal at ~ 5 min) activate ERK1/2. Here, we show that dually-phosphorylated ERK1/2 rapidly (< 2 min) appear in the nucleus following stimulation with endothelin-1. We characterized the active ERK1/2 species in myocytes exposed to endothelin-1 or PMA using MonoQ FPLC. Unexpectedly, two peaks of ERK1 and two peaks of ERK2 activity were resolved using in vitro kinase assays. One of each of these represented the dually-phosphorylated species. The other two represented activities for ERK1 or ERK2 which were phosphorylated solely on the Thr- residue. Monophosphothreonyl ERK1/2 represented maximally ~ 30% of total ERK1/2 activity after stimulation with endothelin-1 or PMA, and their kcat values were estimated to be minimally ~ 30% of the dually-phosphorylated species. Appearance of monophosphothreonyl ERK1/2 was rapid but delayed in comparison with dually-phosphorylated ERK1/2. Of 10 agonists studied, endothelin-1 and PMA were most effective in terms of ERK1/2 activation and in stimulating the appearance of monophosphothreonyl and dually-phosphorylated ERK1/2. Thus, enzymically active monophosphothreonyl ERK1/2 are formed endogenously following activation of the ERK1/2 cascade and we suggest that monophosphothreonyl ERK1/2 arise by protein tyrosine phosphatase-mediated dephosphorylation of dually-phosphorylated ERK1/2.

Use of Megasphaera elsdenii NCIMB41125 as a probiotic for early-lactation dairy cows: effects on rumen pH and fermentation patterns

Multiparous rumen-fistulated Holstein cows were fed, from d 1 to 28 post-calving, an ad libitum TMR containing (g/kg DM) grass silage (196), corn silage (196), wheat (277), soybean meal (100), and other feeds (231) with CP, NDF, starch and water soluble carbohydrate concentrations of 176, 260, 299 and 39 g/kg DM respectively and ME of 12.2 MJ/kg DM. Treatments consisting of a minimum of 1010 cfu Megasphaera elsdenii NCIMB 41125 in 250 ml solution (MEGA) or 250 ml of autoclaved M. elsdenii (CONT) were administered via the rumen cannula on d 3 and 12 of lactation (n=7 per treatment). Mid-rumen pH was measured every 15 minutes and eating and ruminating behavior was recorded for 24 h on d 2, 4, 6, 8, 11, 13, 15, 17, 22 and 28. Rumen fluid for VFA and lactic acid (LA) analysis was collected at 11 timepoints on each of d 2, 4, 6, 13 and 15. Data were analysed as repeated measures using the Glimmix (LA data) or Mixed (all other data) procedures of SAS with previous 305 d milk yield and d 2 measurements as covariates where appropriate. Milk yield was higher (CONT 43.0 vs MEGA 45.4 ±0.75 kg/d, P=0.051) and fat concentration was lower (CONT 45.6 vs MEGA 40.4 ±1.05 g/kg, P=0.005) in cows that received MEGA. Time spent eating (263 ±15 min/d) and ruminating (571 ±13 min/d), DM intake (18.4 ±0.74 kg/d), proportion of each 24 h period with rumen pH below 5.6 (3.69 ±0.94 h) and LA concentrations (2.00 mM) were similar (P>0.327) across treatments. Ruminal total VFA concentration (104 ±3 mM) was similar (P=0.404) across treatments, but a shift from acetate (CONT 551 vs MEGA 524 ±14 mmol/mol VFA, P=0.161) to propionate production (CONT 249 vs MEGA 275 ±11 mmol/mol VFA, P=0.099) meant that the acetate:propionate ratio (CONT 2.33 vs MEGA 1.94 ±0.15) was reduced (P=0.072) in cows that received MEGA. This study provides evidence that supplementation of early lactation dairy cows with MEGA alters rumen fermentation patterns in favour of propionate, with potential benefits for animal health and productivity.

Preparation of (–)-epigallocatechin gallate from commercial green tea by caffeine precipitation and solvent partition

A method has been developed which enables the easy and inexpensive preparation of gram quantities of (–)-epigallocatechin gallate from green tea (Camellia sinensis). A decaffeinated aqueous brew of commercial green tea is treated with caffeine (30 m ). The precipitate is redissolved after decaffeination with chloroform and further purified by solvent partition with ethyl hexanoate and propyl acetate. Commercial leaf (25 g) yields 400 mg (–)-epigallocatechin gallate at better than 80% purity, as judged by reversed phase HPLC.

Self-assembled molecular complexes and coordination polymers of CdII, hexamine, and monocarboxylates: structural analysis and theoretical studies of supramolecular interactions

Four new cadmium(II) complexes [Cd-2(bz)(4)(H2O)(4)(mu 2-hmt)]center dot Hbz center dot H2O (1), [Cd-3(bz)(6)(H2O)(6)(mu 2-hmt)(2)]center dot 6H(2)O (2), [Cd(pa)(2)(H2O)(mu(2)-hmt)](n) (3), and {[Cd-3(ac)(6)(H2O)(3)(mu(3)-hmt)(2)]center dot 6H(2)O}(n) (4) with hexamine (hmt) and monocarboxylate ions, benzoate (bz), phenylacetate (pa), or acetate (ac) have been synthesized and characterized structurally. Structure determinations reveal that 1 is dinuclear, 2 is trinuclear, 3 is a one-dimensional (1D) infinite chain, and 4 is a two-dimensional (2D) polymer with fused hexagonal rings consisting of Cd-II and hmt. All the Cd-II atoms in the four complexes (except one CdII in 2) possess seven-coordinate pentagonal bipyramidal geometry with the various chelating bidentate carboxylate groups in equatorial sites. One of the CdII ions in 2, a complex that contains two monodentate carboxylates is in a distorted octahedral environment. The bridging mode of hmt is mu 2- in complexes 1-3 but is mu 3- in complex 4. In all complexes, there are significant numbers of H-bonds, C-H/pi, and pi-pi interactions which play crucial roles in forming the supramolecular networks. The importance of the noncovalent interactions in terms of energies and geometries has been analyzed using high level ab initio calculations. The effect of the cadmium coordinated to hmt on the energetic features of the C-H/pi interaction is analyzed. Finally, the interplay between C-H/pi and pi-pi interactions observed in the crystal structure of 3 is also studied.

Effect of linseed oil and fish oil alone or as an equal mixture of ruminal fatty acid metabolism in growing steers fed maize silage-based diets

Based on the potential benefits to human health there is interest in increasing 18:3n-3, 20:5n-3, 22:6n-6, and cis-9,trans-11 conjugated linoleic acid (CLA) in ruminant foods. Four Aberdeen Angus steers (406 ± 8.2 kg BW) fitted with rumen and duodenal cannulae were used in a 4 x 4 Latin square experiment with 21 d periods to examine the potential of fish oil (FO) and linseed oil (LO) in the diet to increase ruminal outflow of trans-11 18:1 and total n-3 polyunsaturated fatty acids (PUFA) in growing cattle. Treatments consisted of a control diet (60:40; forage:concentrate ratio, on a DM basis, respectively) based on maize silage, or the same basal ration containing 30 g/kg DM of FO, LO or a mixture (1:1, w/w) of FO and LO (LFO). Diets were offered as total mixed rations and fed at a rate of 85 g DM/kg BW0.75/d. Oils had no effect (P = 0.52) on DM intake. Linseed oil had no effect (P > 0.05) on ruminal pH or VFA concentrations, while FO shifted rumen fermentation towards propionate at the expense of acetate. Compared with the control, LO increased (P < 0.05) 18:0, cis 18:1 (Δ9, 12-15), trans 18:1 (Δ4-9, 11-16), trans 18:2, geometric isomers of ∆9,11, ∆11,13, and ∆13,15 CLA, trans-8,cis-10 CLA, trans-10,trans-12 CLA, trans-12,trans-14 CLA, and 18:3n-3 flow at the duodenum. Inclusion of FO in the diet resulted in higher (P < 0.05) flows of cis-9 16:1, trans 16:1 (Δ6-13), cis 18:1 (Δ9, 11, and 13), trans 18:1 (Δ6-15), trans 18:2, 20:5n-3, 22:5n-3, and 22:6n-3, and lowered (P < 0.001) 18:0 at the duodenum relative to the control. For most fatty acids at the duodenum responses to LFO were intermediate of FO and LO. However, LFO resulted in higher (P = 0.04) flows of total trans 18:1 than LO and increased (P < 0.01) trans-6 16:1 and trans-12 18:1 at the duodenum compared with FO or LO. Biohydrogenation of cis-9 18:1 and 18:2n-6 in the rumen was independent of treatment, but both FO and LO increased (P < 0.001) the extent of 18:3n-3 biohydrogenation compared with the control. Ruminal 18:3n-3 biohydrogenation was higher (P < 0.001) for LO and LFO than FO, while biohydrogenation of 20:5n-3 and 22:6n-3 in the rumen was marginally lower (P = 0.05) for LFO than FO. In conclusion, LO and FO at 30 g/kg DM altered the biohydrogenation of unsaturated fatty acids in the rumen causing an increase in the flow of specific intermediates at the duodenum, but the potential of these oils fed alone or as a mixture to increase n-3 PUFA at the duodenum in cattle appears limited.

Nickel(II) and copper(II) complexes of Schiff base ligands containing N4O2 and N4S2 donors with pyrrole terminal binding groups: synthesis, characterization, X-ray structures, DFT and electrochemical studies

A series of hexadentate ligands, H2Lm (m = 1−4), [1H-pyrrol-2-ylmethylene]{2-[2-(2-{[1H-pyrrol-2-ylmethylene]amino}phenoxy)ethoxy]phenyl}amine (H2L1), [1H-pyrrol-2-ylmethylene]{2-[4-(2-{[1H-pyrrol-2-ylmethylene]amino}phenoxy)butoxy]phenyl}amine (H2L2), [1H-pyrrol-2-ylmethylene][2-({2-[(2-{[1H-pyrrol-2-ylmethylene]amino}phenyl)thio]ethyl}thio)phenyl]amine (H2L3) and [1H-pyrrol-2-ylmethylene][2-({4-[(2-{[1H-pyrrol-2-lmethylene]amino}phenyl)thio]butyl}thio) phenyl]amine (H2L4) were prepared by condensation reaction of pyrrol-2-carboxaldehyde with {2-[2-(2-aminophenoxy)ethoxy]phenyl}amine, {2-[4-(2-aminophenoxy)butoxy]phenyl}amine, [2-({2-[(2-aminophenyl)thio]ethyl}thio)phenyl]amine and [2-({4-[(2-aminophenyl)thio]butyl}thio)phenyl]amine respectively. Reaction of these ligands with nickel(II) and copper(II) acetate gave complexes of the form MLm (m = 1−4), and the synthesized ligands and their complexes have been characterized by a variety of physico-chemical techniques. The solid and solution states investigations show that the complexes are neutral. The molecular structures of NiL3 and CuL2, which have been determined by single crystal X-ray diffraction, indicate that the NiL3 complex has a distorted octahedral coordination environment around the metal while the CuL2 complex has a seesaw coordination geometry. DFT calculations were used to analyse the electronic structure and simulation of the electronic absorption spectrum of the CuL2 complex using TDDFT gives results that are consistent with the measured spectroscopic behavior of the complex. Cyclic voltammetry indicates that all copper complexes are electrochemically inactive but the nickel complexes with softer thioethers are more easily oxidized than their oxygen analogs.

Copper(II) complexes of benzene-1,2-dioxyacetic acid (bdoaH2): X-ray crystal structure of [Cu2(bdoa)(phen)4]bdoa·13H2O (phen = 1,10-phenanthroline)

Copper(II) acetate reacts with benzene-1,2-dioxyacetic acid (bdoaH2) in aqueous media to give [Cu(bdoa)(H2O)2] (1). Complex 1 reacts with the N-donor ligands pyridine (py), ammonia and 1,10-phenanthroline (phen) to give [Cu(bdoa)(NH3)2]·H2O (2), [Cu(bdoa)(py)2]·H2O (3) and [Cu2(bdoa)(phen)4]bdoa·13H2O (4), respectively. The X-ray crystal structure of the dicopper(II,II) complex 4 shows each copper atom at the centre of a distorted trigonal bipyramid comprising four nitrogen atoms from two chelating phen ligands and a single oxygen atom from one of the carboxylate moieties of the bridging bdoa2− ligand. The cyclic voltammogram of 4 shows a single reversible wave for the Cu2+/Cu+ couple at E = + 115 mV (vs Ag/AgCl). Spectroscopic and magnetic data for the complexes are given.

Synthesis and properties of tetra-μ-acetatodiruthenium(II,III) phenylphosphinate and phenylphosphonate complexes: X-ray crystal structures of [Ru2(μ-O2CCH3)4(HPhPO2)2]H and [Ru2(μ-O2CCH3)4(PhPO3H)2]H·H2O

Phenylphosphinic acid (HPhPO2H) and phenylphosphonic acid (PhPO3H2) react with a methanolic solution of [Ru2(μ-O2CCH3)4(O2CCH3)2]H·0.7H2O at room temperature to give [Ru2(μ-O2CCH3)4(HPhPO2)2H (1) and [Ru2(μ-O2CCH3)4 (PhPO3H)2]H·H2O (2), respectively. The X-ray crystal structures of 1 and 2 each show the RuRu core to be ligated by four bridging bidentate acetate ligands [RuRu distances: 1 = 2.272(1) Å; 2 = 2.267(2) Å] and two axial phenylphosphinate and phenylphosphonate ligands, respectively. In each complex the individual bimetallic molecules are linked together by a hydrogen ion which bridges the oxygen atoms of neighbouring axial ligands. In 2 the water molecule is also hydrogen-bonded to one of the axial phenylphosphonate groups. Spectroscopic, magnetic and cyclic voltammetric data for the complexes are given.