Metformin selectively targets redox control of complex I energy transduction.

Many guanide-containing drugs are antihyperglycaemic but most exhibit toxicity, to the extent that only the biguanide metformin has enjoyed sustained clinical use. Here, we have isolated unique mitochondrial redox control properties of metformin that are likely to account for this difference. In primary hepatocytes and H4IIE hepatoma cells we found that antihyperglycaemic diguanides DG5-DG10 and the biguanide phenformin were up to 1000-fold more potent than metformin on cell signalling responses, gluconeogenic promoter expression and hepatocyte glucose production.

Investigation of salicylate hepatic responses in comparison with chemical analogues of the drug.

Anti-hyperglycaemic effects of the hydroxybenzoic acid salicylate might stem from effects of the drug on mitochondrial uncoupling, activation of AMP-activated protein kinase, and inhibition of NF-κB signalling. Here, we have gauged the contribution of these effects to control of hepatocyte glucose production, comparing salicylate with inactive hydroxybenzoic acid analogues of the drug. In rat H4IIE hepatoma cells, salicylate was the only drug tested that activated AMPK.

Cellular responses to the metal-binding properties of metformin.

In recent decades, the antihyperglycemic biguanide metformin has been used extensively in the treatment of type 2 diabetes, despite continuing uncertainty over its direct target. In this article, using two independent approaches, we demonstrate that cellular actions of metformin are disrupted by interference with its metal-binding properties, which have been known for over a century but little studied by biologists. We demonstrate that copper sequestration opposes known actions of metformin not only on AMP-activated protein kinase (AMPK)-dependent signaling, but also on S6 protein phosphorylation.

The anti-neurodegenerative agent clioquinol regulates the transcription factor FOXO1a.

Many diseases of aging including AD (Alzheimer's disease) and T2D (Type 2 diabetes) are strongly associated with common risk factors, suggesting that there may be shared aging mechanisms underlying these diseases, with the scope to identify common cellular targets for therapy. In the present study we have examined the insulin-like signalling properties of an experimental AD 8-hydroxyquinoline drug known as CQ (clioquinol). The IIS [insulin/IGF-1 (insulin-like growth factor-1) signalling] kinase Akt/PKB (protein kinase B) inhibits the transcription factor FOXO1a (forkhead box O1a) by phosphorylating it on residues that trigger its exit from the nucleus.

Zinc-dependent effects of small molecules on the insulin-sensitive transcription factor FOXO1a and gluconeogenic genes.

Metal-binding compounds have recently been reported to have anti-hyperglycaemic properties in vivo. In the current study, we have investigated the ability of these compounds and related structures to induce insulin-like signal transduction to downstream effectors such as the transcription factor FOXO1a and the key gluconeogenic regulatory enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase). Our results indicate that β-thujaplicin, diethyldithiocarbamate (DEDTC) and its clinically-used dimer disulfiram, induce insulin-like dose-dependent effects on signalling to FOXO1a in a manner that is strictly dependent on the presence of zinc ions, as other ions including aluminium, cobalt, copper, lithium and manganese cannot substitute.

Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR.

AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation.

Phosphorylation and 14-3-3 binding of Arabidopsis trehalose-phosphate synthase 5 in response to 2-deoxyglucose.

Trehalose-6-phosphate is a 'sugar signal' that regulates plant metabolism and development. The Arabidopsis genome encodes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphatase (TPP) enzymes. It also encodes class II proteins (TPS isoforms 5-11) that contain both TPS-like and TPP-like domains, although whether these have enzymatic activity is unknown.

Phosphorylation and 14-3-3 binding of Arabidopsis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Fructose 2,6-bisphosphate (fru-2,6-P2) is a signalling metabolite that regulates photosynthetic carbon partitioning in plants. The content of fru-2,6-P2 in Arabidopsis leaves varied in response to photosynthetic activity with an abrupt decrease at the start of the photoperiod, gradual increase through the day, and modest decrease at the start of the dark period. In Arabidopsis suspension cells, fru-2,6-P2 content increased in response to an unknown signal upon transfer to fresh culture medium.

Regulation of the 14-3-3-binding protein p39 by growth factors and nutrients in rat PC12 pheochromocytoma cells.

Unstimulated PC12 pheochromocytoma cells contain many proteins that bound to 14-3-3s in competition with a 14-3-3-binding peptide. Additional proteins, including one of 39 kDa (p39), became capable of binding to 14-3-3s in phosphatidylinositol 3-kinase-dependent responses to epidermal growth factor or nerve growth factor in vivo. The growth factor regulation was unaffected by inhibitors of the mitogen- or stress-activated protein kinase pathways, or by glucose starvation, but was blocked by amino acid starvation and only partially blocked by rapamycin.

Affinity purification of diverse plant and human 14-3-3-binding partners.

Many proteins that bind to a 14-3-3 column in competition with a 14-3-3-binding phosphopeptide have been purified from plant and mammalian cells and tissues. New 14-3-3 targets include enzymes of biosynthetic metabolism, vesicle trafficking, cell signalling and chromatin function. These findings indicate central regulatory roles for 14-3-3s in partitioning carbon among the pathways of sugar, amino acid, nucleotide and protein biosynthesis in plants.

Phosphorylation-dependent interactions between enzymes of plant metabolism and 14-3-3 proteins.

Far-Western overlays of soluble extracts of cauliflower revealed many proteins that bound to digoxygenin (DIG)-labelled 14-3-3 proteins. Binding to DIG-14-3-3s was prevented by prior dephosphorylation of the extract proteins or by competition with 14-3-3-binding phosphopeptides, indicating that the 14-3-3 proteins bind to phosphorylated sites. The proteins that bound to the DIG-14-3-3s were also immunoprecipitated from extracts with anti-14-3-3 antibodies, demonstrating that they were bound to endogenous plant 14-3-3 proteins.

Core alpha1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts.

Carbohydrates have been suggested to account for some IgE cross-reactions between various plant, insect, and mollusk extracts, while some IgG antibodies have been successfully raised against plant glycoproteins. A rat monoclonal antibody raised against elderberry abscission tissue (YZ1/2.23) and rabbit polyclonal antiserum against horseradish peroxidase were screened for reactivity in enzyme-linked immunosorbent assay against a range of plant glycoproteins and extracts as well as neoglycoproteins, bee venom phospholipase, and several animal glycoproteins.

Glycan modification of a thermostable recombinant (1-3, 1-4)-beta-glucanase secreted from Saccharomyces cerevisiae is determined by strain and culture conditions.

High level biosynthesis and secretion of the thermostable hybrid (1-3, 1-4)- beta-glucanase H(A16-M) has been achieved in Saccharomyces cerevisiae by means of the yeast vacuolar endoprotease B promoter (PRB1P) and the Bacillus macerans (1-3, 1-4)-beta-glucanase signal peptide. The N-glycans present on the yeast-secreted H(A16-M), denoted H(A16-M)-Y, were released by endoglycosidase H, and identified by proton NMR spectroscopy to be a homologous series of Man8-13GlcNAc2, although only traces of Man9GlcNAc2 were found. Therefore, processing of N-glycans on H(A16-M)-Y is similar to that on homologous proteins.

cDNA, amino acid and carbohydrate sequence of barley seed-specific peroxidase BP 1.

The major peroxidase of barley seed BP 1 was characterized. Previous studies showed a low carbohydrate content, low specific activity and tissue-specific expression, and suggested that this basic peroxidase could be particularly useful in the elucidation of the structure-function relationship and in the study of the biological roles of plant peroxidases (S.K.

Structure-activity in the carrageenans: iota-carrageenan and experimental oedemagenic activity.

The oedemagenic activities of several iota-carrageenans from Eucheuma spinosum have been compared using the rat hindpaw model. From the parent iota-carrageenan of weight average molecular weight 541 100 an acid-degraded iota-carrageenan (Mw = 20 300) was obtained which on fractionation yielded five iota-carrageenans Mw ranging from 73 700 to 4 600. The oedemagenic activity of iota-carrageenan resided in the fraction of Mw = 73 700, the parent undegraded high molecular weight iota-carrageenan being no more active and fractions of lower molecular weight being inactive.

The macroanionic activity of heparins in the presence of dextrose and calcium ion.

The effect of dextrose on heparin was investigated using the heparin-azur A interaction as a measure of macroanionic activity. Dextrose solutions did not diminish heparin-azur A metachromasia but prior autoclaving of the dextrose solution resulted in a slight decrease. When calcium chloride was added to the dextrose (autoclaved or unautoclaved) there was a reduction which was greater than that caused by calcium ion in the absence of dextrose.

The anticoagulant activity of heparins in dextrose solutions.

Changes with time in the anti-factor Xa activity of several heparins were determined in freshly autoclaved and unautoclaved dextrose solutions. In the former, activity was raised, in the latter a reversible fall occurred at certain heparin concentrations, and the term "dextrose effect' is applied to the difference in activity in the two types of dextrose solutions. A simple dependence on heparin concentration for the rise in activity in autoclaved dextrose solutions contrasted with a threshold heparin concentration in the unautoclaved dextrose solutions.

Barium sulphate preparations for use in double contrast examination of the upper gastrointestinal tract.

Physical properties relevant to upper gastrointestinal radiology have been compared for five barium sulphate preparations and related to radiographic results. Evaluation of particles (size and stability) and whole suspension (dispersibility and fluidity) resulted in ranking of the preparations generally in accord with that based on radiological experience in double contrast examinations of the stomach. Experiments with extirpated pig stomach revealed a tendency for large particles in a low viscosity barium sulphate suspension to settle in mucosal grooves.

The influence of molecular size and pH on the macrocationic inhibition of pepsin by polylysine.

Polylysines of 7-371 lysine residues inhibited pepsin over the pH range 3.6-5.0 in a system using azocoll as substrate.