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.

Inactivation of the antibacterial and cytotoxic properties of silver ions by biologically relevant compounds

There has been a recent surge in the use of silver as an antimicrobial agent in a wide range of domestic and clinical products, intended to prevent or treat bacterial infections and reduce bacterial colonization of surfaces. It has been reported that the antibacterial and cytotoxic properties of silver are affected by the assay conditions, particularly the type of growth media used in vitro. The toxicity of Ag+ to bacterial cells is comparable to that of human cells. We demonstrate that biologically relevant compounds such as glutathione, cysteine and human blood components significantly reduce the toxicity of silver ions to clinically relevant pathogenic bacteria and primary human dermal fibroblasts (skin cells). Bacteria are able to grow normally in the presence of silver nitrate at >20-fold the minimum inhibitory concentration (MIC) if Ag+ and thiols are added in a 1:1 ratio because the reaction of Ag+ with extracellular thiols prevents silver ions from interacting with cells. Extracellular thiols and human serum also significantly reduce the antimicrobial activity of silver wound dressings Aquacel-Ag (Convatec) and Acticoat (Smith & Nephew) to Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli in vitro. These results have important implications for the deployment of silver as an antimicrobial agent in environments exposed to biological tissue or secretions. Significant amounts of money and effort have been directed at the development of silver-coated medical devices (e.g. dressings, catheters, implants). We believe our findings are essential for the effective design and testing of antimicrobial silver coatings.

Enterolactone restricts the proliferation of the LNCaP human prostate cancer cell line in vitro

Ecological data suggest a long-term diet high in plant material rich in biologically active compounds, such as the lignans, can significantly influence the development of prostate cancer over the lifetime of an individual. The capacity of a pure mammalian lignan, enterolactone (ENL), to influence the proliferation of the LNCaP human prostate cancer cell line was investigated as a function of cell density, metabolic activity, expression and secretion of prostate specific antigen (PSA), cell cycle profile, and the expression of genes involved in development and progression of prostate cancer. Treatment with a subcytotoxic concentration of ENL (60 mu M for 72 h) was found to reduce: cell density (57.5%, SD 7.23, p < 0.001), metabolic activity (55%, SD 0.03, p < 0.001), secretion of PSA (48.50% SD 4.74, p = 0.05) and induce apoptosis (8.33-fold SD 0.04, p = 0.001) compared to untreated cells. Cotreatment with 10 mu M etoposide was found to increase apoptosis by 50.17% (SD 0.02, p < 0.001). Additionally, several key genes (e.g. MCMs, survivin and CDKs) were beneficially regulated by ENL treatment (p < 0.05). The data suggest that the antiproliferative activity of ENL is a consequence of altered expression of cell cycle associated genes and provides novel molecular evidence for the antiproliferative properties of a pure lignan in prostate cancer.

The gut microbiota and lipid metabolism: implications for human health and coronary heart disease

Coronary heart disease (CHD) is the leading cause of mortality in Western societies, affecting about one third of the population before their seventieth year. Over the past decades modifiable risk factors of CHD have been identified, including smoking and diet. These factors when altered can have a significant impact on an individuals' risk of developing CHD, their overall health and quality of life. There is strong evidence suggesting that dietary intake of plant foods rich in fibre and polyphenolic compounds, effectively lowers the risk of developing CHD. However, the efficacy of these foods often appears to be greater than the sum of their recognised biologically active parts. Here we discuss the hypothesis that beneficial metabolic and vascular effects of dietary fibre and plant polyphenols are due to an up regulation of the colon-systemic metabolic axis by these compounds. Fibres and many polyphenols are converted into biologically active compounds by the colonic microbiota. This microbiota imparts great metabolic versatility and dynamism, with many of their reductive or hydrolytic activities appearing complementary to oxidative or conjugative human metabolism. Understanding these microbial activities is central to determining the role of different dietary components in preventing or beneficially impacting on the impaired lipid metabolism and vascular dysfunction that typifies CHD and type 11 diabetes. This approach lays the foundation for rational selection of health promoting foods, rational target driven design of functional foods, and provides an essential thus-far, overlooked, dynamic to our understanding of how foods recognised as "healthy" impact on the human metabonome.

Apples and cardiovascular health: is the gut microbiota a core consideration?

There is now considerable scientific evidence that a diet rich in fruits and vegetables can improve human health and protect against chronic diseases. However, it is not clear whether different fruits and vegetables have distinct beneficial effects. Apples are among the most frequently consumed fruits and a rich source of polyphenols and fiber. A major proportion of the bioactive components in apples, including the high molecular weight polyphenols, escape absorption in the upper gastrointestinal tract and reach the large intestine relatively intact. There, they can be converted by the colonic microbiota to bioavailable and biologically active compounds with systemic effects, in addition to modulating microbial composition. Epidemiological studies have identified associations between frequent apple consumption and reduced risk of chronic diseases such as cardiovascular disease. Human and animal intervention studies demonstrate beneficial effects on lipid metabolism, vascular function and inflammation but only a few studies have attempted to link these mechanistically with the gut microbiota. This review will focus on the reciprocal interaction between apple components and the gut microbiota, the potential link to cardiovascular health and the possible mechanisms of action.

Metabolism of Maillard reaction products by the human gut microbiota - implications for health

The human colonic microbiota imparts metabolic versatility on the colon, interacts at many levels in healthy intestinal and systemic metabolism, and plays protective roles in chronic disease and acute infection. Colonic bacterial metabolism is largely dependant on dietary residues from the upper gut. Carbohydrates, resistant to digestion, drive colonic bacterial fermentation and the resulting end products are considered beneficial. Many colonic species ferment proteins but the end products are not always beneficial and include toxic compounds, such as amines and phenols. Most components of a typical Western diet are heat processed. The Maillard reaction, involving food protein and sugar, is a complex network of reactions occurring during thermal processing. The resultant modified protein resists digestion in the small intestine but is available for colonic bacterial fermentation. Little is known about the fate of the modified protein but some Maillard reaction products (MRP) are biologically active by, e.g. altering bacterial population levels within the colon or, upon absorption, interacting with human disease mechanisms by induction of inflammatory responses. This review presents current understanding of the interactions between MRP and intestinal bacteria. Recent scientific advances offering the possibility of elucidating the consequences of microbe-MRP interactions within the gut are discussed.

Metabolism of Maillard reaction products by the human gut microbiota: implications for health

The human colonic microbiota imparts metabolic versatility on the colon, interacts at many levels in healthy intestinal and systemic metabolism, and plays protective roles in chronic disease and acute infection. Colonic bacterial metabolism is largely dependant on dietary residues from the upper gut. Carbohydrates, resistant to digestion, drive colonic bacterial fermentation and the resulting end products are considered beneficial. Many colonic species ferment proteins but the end products are not always beneficial and include toxic compounds, such as amines and phenols. Most components of a typical Western diet are heat processed. The Maillard reaction, involving food protein and sugar, is a complex network of reactions occurring during thermal processing. The resultant modified protein resists digestion in the small intestine but is available for colonic bacterial fermentation. Little is known about the fate of the modified protein but some Maillard reaction products (MRP) are biologically active by, e.g. altering bacterial population levels within the colon or, upon absorption, interacting with human disease mechanisms by induction of inflammatory responses. This review presents current understanding of the interactions between MRP and intestinal bacteria. Recent scientific advances offering the possibility of elucidating the consequences of microbe-MRP interactions within the gut are discussed.

Methylxanthines are the psycho-pharmacologically active constituents of chocolate

Rationale: Liking, cravings and addiction for chocolate ("chocoholism") are often explained through the presence of pharmacologically active compounds. However, mere "presence" does not guarantee psycho-activity. Objectives: Two double-blind, placebo-controlled studies measured the effects on cognitive performance and mood of the amounts of cocoa powder and methylxanthines found in a 50 g bar of dark chocolate. Methods: In study 1, participants (n=20) completed a test battery once before and twice after treatment administration. Treatments included 11.6 g cocoa powder and a caffeine and theobromine combination (19 and 250 mg, respectively). Study 2 (n=22) comprised three post-treatment test batteries and investigated the effects of "milk" and "dark" chocolate levels of these methylxanthines. The test battery consisted of a long duration simple reaction time task, a rapid visual information processing task, and a mood questionnaire. Results: Identical improvements on the mood construct "energetic arousal" and cognitive function were found for cocoa powder and the caffeine+theobromine combination versus placebo. In chocolate, both "milk chocolate" and "dark chocolate" methylxanthine doses improved cognitive function compared with "white chocolate". The effects of white chocolate did not differ significantly from those of water. Conclusion: A normal portion of chocolate exhibits psychopharmacological activity. The identical profile of effects exerted by cocoa powder and its methylxanthine constituents shows this activity to be confined to the combination of caffeine and theobromine. Methylxanthines may contribute to the popularity of chocolate; however, other attributes are probably much more important in determining chocolate's special appeal and in explaining related self-reports of chocolate cravings and "chocoholism".

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.

Global warming potentials and radiative efficiencies of halocarbons and related compounds: a comprehensive review

In the mid-1970s it was recognized that, as well as being substances that deplete stratospheric ozone, chlorofluorocarbons (CFCs) were strong greenhouse gases that could have substantial impacts on radiative forcing of climate change. Around a decade later, this group of radiatively active compounds was expanded to include a large number of replacements for ozone-depleting substances such as chlorocarbons, hydrochlorocarbons, hydrochlorofluorocarbons (HCFCs), hydrofluorocarbons (HFCs), perfluorocarbons (PFCs), bromofluorocarbons, and bromochlorofluorocarbons. This paper systematically reviews the published literature concerning the radiative efficiencies (REs) of CFCs, bromofluorocarbons and bromochlorofluorocarbons (halons), HCFCs, HFCs, PFCs, SF6, NF3, and related halogen containing compounds. In addition we provide a comprehensive and self-consistent set of new calculations of REs and global warming potentials (GWPs) for these compounds, mostly employing atmospheric lifetimes taken from the available literature. We also present Global Temperature change Potentials (GTPs) for selected gases. Infrared absorption spectra used in the RE calculations were taken from databases and individual studies, and from experimental and ab initio computational studies. Evaluations of REs and GWPs are presented for more than 200 compounds. Our calculations yield REs significantly (> 5%) different from those in the Intergovernmental Panel on Climate Change Fourth Assessment Report (AR4) for 49 compounds. We present new RE values for more than 100 gases which were not included in AR4. A widely-used simple method to calculate REs and GWPs from absorption spectra and atmospheric lifetimes is assessed and updated. This is the most comprehensive review of the radiative efficiencies and global warming potentials of halogenated compounds performed to date.

Exploring quercetin and luteolin derivatives as antiangiogenic agents

The formation of new blood vessels from the pre-existing vasculature (angiogenesis) is a crucial stage in cancer progression and, indeed, angiogenesis inhibitors are now used as anticancer agents, clinically. Here we have explored the potential of flavonoid derivatives as antiangiogenic agents. Specifically, we have synthesised methoxy and 4-thio derivatives of the natural flavones quercetin and luteolin, two of which (4-thio quercetin and 4-thio luteolin) had never been previously reported. Seven of these compounds showed significant (P<0.05) antiangiogenic activity in an in vitro scratch assay. Their activity ranged from an 86% inhibition of the vascular endothelium growth factor (VEGF)-stimulated migration (observed for methoxyquercetin at 10 µM and for luteolin at 1 µM) to a 36% inhibition (for thiomethoxy quercetin at 10 µM). Western blotting studies showed that most (4 out of 7) compounds inhibited phosphorylation of the VEGF receptor-2 (VEGFR2), suggesting that the antiangiogenic activity was due to an interference with the VEGF/VEGFR2 pathway. Molecular modelling studies looking at the affinity of our compounds towards VEGFR and/or VEGF confirmed this hypothesis, and indeed the compound with the highest antiangiogenic activity (methoxyquercetin) showed the highest affinity towards VEGFR and VEGF. As reports from others have suggested that structurally similar compounds can elicit biological responses via a non-specific, promiscuous membrane perturbation, potential interactions of the active compounds with a model lipid bilayer were assessed via DSC. Luteolin and its derivatives did not perturb the model membrane even at concentrations 10 times higher than the biologically active concentration and only subtle interactions were observed for quercetin and its derivatives. Finally, cytotoxicity assessment of these flavonoid derivatives against MCF-7 breast cancer cells demonstrated also a direct anticancer activity albeit at generally higher concentrations than those required for an antiangiogenic effect (10 fold higher for the methoxy analogues). Taken together these results show promise for flavonoid derivatives as antiangiogenic agents.

Systemic and persistent effect of neem (Azadirachta indica) formulations against root-knot nematodes, Meloidogyne javanica and their storage life

Neem leaves, neem cake (a by-product left after the extraction of oil from neem seed) and a commercially refined product aza (azadirachtin) extracted from seed were evaluated. Aqueous extracts of crude neem formulations used as a seedling dip treatment significantly reduced the number of females and egg masses in roots whereas the refined one did not. A split-root technique was used to demonstrate the translocation of active compounds within a plant and their subsequent effect on the development of nematodes. When applied to the root portion all formulations significantly reduced the number of egg masses and eggs per egg mass. Whereas on the untreated root portion, neem cake at 3% w/w and aza at 0.1% w/w significantly reduced the number of egg masses as compared with neem leaves at 3% w/w, aza at 0.05% and control. All the neern formulations significantly reduced the number of eggs per egg mass on' the untreated root portion. The effect of neem leaves and cake on the development of root-knot nematodes was tested at 2, 4, 6, 8, and 16 weeks after their application to soil. Even after 16 weeks all the treatments significantly reduced the galling index and number of egg masses but their effectiveness declined over time. After storing neem leaves, cake and aza for 8 months under ambient conditions the efficacy of neem leaves and aza, against root-knot nematodes, remained stable whereas that of cake declined. (c) 2006 Elsevier Ltd. All rights reserved.

The tissue-specific processing of pro-opiomelanocortin

It is just over 30 years since the definitive identification of the adrenocorticotrophin (ACTH) precursor, pro-opiomelanocotin (POMC). Although first characterised in the anterior and intermediate lobes of the pituitary, POMC is also expressed in a number of both central and peripheral tissues including the skin, central nervous tissue and placenta. Following synthesis, POMC undergoes extensive post-translational processing producing not only ACTH, but also a number of other biologically active peptides. The extent and pattern of this processing is tissue-specific, the end result being the tissue dependent production of different combinations of peptides from the same precursor. These peptides have a diverse range of biological roles ranging from pigmentation to adrenal function to the regulation of feeding. This level of complexity has resulted in POMC becoming the archetypal model for prohormone processing, illustrating how a single protein combined with post-translational modification can have a diverse number of roles.

Outlining eicosanoid biosynthesis in the crustacean Daphnia

Background: Eicosanoids are biologically active, oxygenated metabolites of three C20 polyunsaturated fatty acids. They act as signalling molecules within the autocrine or paracrine system in both vertebrates and invertebrates mainly functioning as important mediators in reproduction, the immune system and ion transport. The biosynthesis of eicosanoids has been intensively studied in mammals and it is known that they are synthesised from the fatty acid, arachidonic acid, through either the cyclooxygenase (COX) pathway; the lipoxygenase (LOX) pathway; or the cytochrome P450 epoxygenase pathway. However, little is still known about the synthesis and structure of the pathway in invertebrates. Results: Here, we show transcriptomic evidence from Daphnia magna (Crustacea: Branchiopoda) together with a bioinformatic analysis of the D. pulex genome providing insight on the role of eicosanoids in these crustaceans as well as outlining a putative pathway of eicosanoid biosynthesis. Daphnia appear only to have one copy of the gene encoding the key enzyme COX, and phylogenetic analysis reveals that the predicted protein sequence of Daphnia COX clusters with other invertebrates. There is no current evidence of an epoxygenase pathway in Daphnia; however, LOX products are most certainly synthesised in daphnids. Conclusion: We have outlined the structure of eicosanoid biosynthesis in Daphnia, a key genus in freshwater ecosystems. Improved knowledge of the function and synthesis of eicosanoids in Daphnia and other invertebrates could have important implications for several areas within ecology. This provisional overview of daphnid eicosanoid biosynthesis provides a guide on where to focus future research activities in this area.

Improved display of synthetic IgG-binding domains on the baculovirus surface

Improved display of foreign protein moieties in combination with beneficial alteration of the viral surface properties should be of value for targeted and enhanced gene delivery. Here, we describe a vector based on Autographa californica multiple nucleopolyhedrovirus (AcMNPV) displaying synthetic IgG-bincling domains (ZZ) of protein A fused to the transmembrane anchor of vesicular stomatitis virus (VSV) G protein. This display vector was equipped with a GFP/EGFP expression cassette enabling fluorescent detection in both insect and mammalian cells. The virus construct displayed the biologically active fusion protein efficiently and showed increased binding capacity to IgG. As the display is carried out using a membrane anchor of foreign origin, gp64 is left intact for virus entry, which may increase gene expression in the transduced mammalian cells. In addition, the viral vector can be targeted to any desired cell type via binding of ZZ domains when an appropriate IgG antibody is available.