Epinephrine-induced hyperpolarization of islet cells without KATP channels

ID-1101 (4-hydroxyisoleucine), an amino acid extracted from fenugreek seeds, exhibits an interesting glucose-dependent insulin-stimulating activity. The present study was undertaken to investigate a possible extrapancreatic effect of ID-1101 on insulin signaling and action besides its previously described insulinotropic action. Insulin-sensitizing effects of ID-1101 were investigated in rat in vivo by three different approaches: 1) using euglycemic hyperinsulinemic clamps in two different rat models of insulin resistance, i.e., Zucker fa/fa rats and rats fed a sucrose-lipid diet; 2) measuring liver and muscle phosphatidylinositol (PI) 3-kinase activity after an acute injection of ID-1101 in normal and insulin-resistant diabetic rats; and 3) after chronic treatment in two rat models of insulin resistance. Euglycemic hyperinsulinemic clamp experiments revealed that ID-1101 can improve insulin resistance through an increase of peripheral glucose utilization rate in sucrose-lipid-fed rats and by decreasing hepatic glucose production in Zucker fa/fa rats. Moreover, we demonstrated that a single injection of ID-1101 activates the PI 3-kinase activity in liver and muscle from normal rats but also in muscle from diabetic rats. Finally, chronic ID-1101 treatment significantly reduced insulinemia in type 2 diabetic rats and reduced the progression of hyperinsulinemia in insulin-resistant obese Zucker fa/fa rats. These findings clearly demonstrate that ID-1101 can reduce insulin resistance through activation of the early steps of insulin signaling in peripheral tissues and in liver. In summary, ID-1101, besides its insulinotropic effect, directly improves insulin sensitivity, making it a potentially very valuable therapeutic agent for diabetes treatment.     

Meridianins, a new family of protein kinase inhibitors isolated from the ascidian Aplidium meridianum

Meridianins are brominated 3-(2-aminopyrimidine)-indoles which are purified from Aplidium meridianum, an Ascidian from the South Atlantic (South Georgia Islands). We here show that meridianins inhibit various protein kinases such as cyclin-dependent kinases, glycogen synthase kinase-3, cyclic nucleotide-dependent kinases and casein kinase 1. Meridianins prevent cell proliferation and induce apoptosis, a demonstration of their ability to enter cells and to interfere with the activity of kinases important for cell division and cell death. These results suggest that meridianins constitute a promising scaffold from which more potent and selective protein kinase inhibitors could be designed.     

Ext1-dependent heparan sulfate regulates the range of Ihh signaling during endochondral ossification

Exostosin1 (Ext1) belongs to a family of glycosyltransferases necessary for the synthesis of the heparan sulfate (HS) chains of proteoglycans, which regulate signaling of several growth factors. Loss of tout velu (ttv), the homolog of Ext1 in Drosophila, inhibits Hedgehog movement. In contrast, we show that reduced HS synthesis in mice carrying a hypomorphic mutation in Ext1 results in an elevated range of Indian hedgehog (Ihh) signaling during embryonic chondrocyte differentiation. Our data suggest a dual function for HS: First, HS is necessary to bind Hedgehog in the extracellular space. Second, HS negatively regulates the range of Hedgehog signaling in a concentration-dependent manner. Additionally, our data indicate that Ihh acts as a long-range morphogen, directly activating the expression of parathyroid hormone-like hormone. Finally, we propose that the development of exostoses in the human Hereditary Multiple Exostoses syndrome can be attributed to activation of Ihh signaling.     

Annotating nucleic acid-binding function based on protein structure

Many of the targets of structural genomics will be proteins with little or no structural similarity to those currently in the database. Therefore, novel function prediction methods that do not rely on sequence or fold similarity to other known proteins are needed. We present an automated approach to predict nucleic-acid-binding (NA-binding) proteins, specifically DNA-binding proteins. The method is based on characterizing the structural and sequence properties of large, positively charged electrostatic patches on DNA-binding protein surfaces, which typically coincide with the DNA-binding-sites. Using an ensemble of features extracted from these electrostatic patches, we predict DNA-binding proteins with high accuracy. We show that our method does not rely on sequence or structure homology and is capable of predicting proteins of novel-binding motifs and protein structures solved in an unbound state. Our method can also distinguish NA-binding proteins from other proteins that have similar, large positive electrostatic patches on their surfaces, but that do not bind nucleic acids.     

Two uncommon phospholipase D isoenzymes from poppy seedlings (Papaver somniferum L.)

Phospholipase D (PLD) has been detected in seedlings of Papaver somniferum L. cv. Lazúr (Papaveraceae). Purification of the enzyme revealed the existence of two forms of PLD (named as PLD-A and PLD-B). The two enzymes strongly differ in their catalytic properties. The pH optima were found at pH 8.0 for PLD-A and at pH 5.5 for PLD-B. While both enzymes show hydrolytic activity toward phosphatidylcholine (PC) and phosphatidyl-p-nitrophenol (PpNP), PLD-B only was able to catalyze the exchange of choline in PC by glycerol. Both enzymes were activated by Ca(2+) ions with an optimum concentration of 10 mM. In contrast to PLDs from other plants, PLD-B was still more activated by Zn(2+) ions with an optimum concentration of 5 mM. The apparent molecular masses of PLD-A and PLD-B, derived from sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), were estimated to be 116.4 and 114.1 kDa. N-terminal protein sequencing indicated N-terminal blockage in both cases. The isoelectric points were found to be 8.7 for PLD-A and 6.7 for PLD-B. Both enzymes were shown to be N-linked glycoproteins. This paper is the first report on PLD in poppy and indicates some important differences of the two enzyme forms to other PLDs known so far.     

Laminin alpha5 chain is required for intestinal smooth muscle development

Laminins (comprised of alpha, beta, and gamma chains) are heterotrimeric glycoproteins integral to all basement membranes. The function of the laminin alpha5 chain in the developing intestine was defined by analysing laminin alpha5(-/-) mutants and by grafting experiments. We show that laminin alpha5 plays a major role in smooth muscle organisation and differentiation, as excessive folding of intestinal loops and delay in the expression of specific markers are observed in laminin alpha5(-/-) mice. In the subepithelial basement membrane, loss of alpha5 expression was paralleled by ectopic or accelerated deposition of laminin alpha2 and alpha4 chains; this may explain why no obvious defects were observed in the villous form and enterocytic differentiation. This compensation process is attributable to mesenchyme-derived molecules as assessed by chick/mouse alpha5(-/-) grafted associations. Lack of the laminin alpha5 chain was accompanied by a decrease in epithelial alpha3beta1 integrin receptor expression adjacent to the epithelial basement membrane and of Lutheran blood group glycoprotein in the smooth muscle cells, indicating that these receptors are likely mediating interactions with laminin alpha5-containing molecules. Taken together, the data indicate that the laminin alpha5 chain is essential for normal development of the intestinal smooth muscle and point to possible mesenchyme-derived compensation to promote normal intestinal morphogenesis when laminin alpha5 is absent.     

Role for laminin-alpha5 chain LG4 module in epithelial branching morphogenesis

Laminin-alpha5 chain was localized in all epithelial basement membranes (BMs) of mouse submandibular gland (SMG) from the onset of branching morphogenesis and became restricted to BMs of epithelial ducts in the adult. To investigate whether the laminin-alpha5 chain plays a role in branching morphogenesis, a set of cell-adhesive peptides from the C-terminal globular domains (LG1-5) was tested for their effects in SMG organ cultures. One peptide, LVLFLNHGH (A5G77f), which represents a sequence located in the connecting loop between strands E and F of LG4, perturbed branching morphogenesis and resulted in irregularities in the contours of epithelial structures, with formation of deep clefts. The data suggest a role for the laminin-alpha5 LG4 module in the development of the duct system, rather than in the bifurcation of epithelial clusters. The epithelial BM of A5G77f-peptide-treated explants was continuous, which was in contrast to our previous finding of impaired epithelial BM assembly in explants treated with the laminin-alpha1 LG4 module peptide, or with a monoclonal antibody against this domain. A5G77f also perturbed in vitro development of lung and kidney. These results suggest a crucial role for the LG4 module of laminin-alpha5 in epithelial morphogenesis that is distinct from that of the laminin-alpha1 LG4.     

Crystal structure of a statin bound to a class II hydroxymethylglutaryl-CoA reductase

Hydroxymethylglutaryl-CoA (HMG-CoA) reductase is the primary target in the current clinical treatment of hypercholesterolemias with specific inhibitors of the "statin" family. Statins are excellent inhibitors of the class I (human) enzyme but relatively poor inhibitors of the class II enzymes of important bacterial pathogens. To investigate the molecular basis for this difference we determined the x-ray structure of the class II Pseudomonas mevalonii HMG-CoA reductase in complex with the statin drug lovastatin. The structure shows lovastatin bound in the active site and its interactions with residues critically involved in catalysis and substrate binding. Binding of lovastatin also displaces the flap domain of the enzyme, which contains the catalytic residue His-381. Comparison with the structures of statins bound to the human enzyme revealed a similar mode of binding but marked differences in specific interactions that account for the observed differences in affinity. We suggest that these differences might be exploited to develop selective class II inhibitors for use as antibacterial agents against pathogenic microorganisms.     

Mechanism of sulfide-quinone reductase investigated using site-directed mutagenesis and sulfur analysis

Biological sulfide oxidation is a reaction occurring in all three domains of life. One enzyme responsible for this reaction in many bacteria has been identified as sulfide:quinone oxidoreductase (SQR). The enzyme from Rhodobacter capsulatus is a peripherally membrane-bound flavoprotein with a molecular mass of approximately 48 kDa, presumably acting as a homodimer. In this work, SQR from Rb. capsulatus has been modified with an N-terminal His tag and heterologously expressed in and purified from Escherichia coli. Three cysteine residues have been shown to be essential for the reductive half-reaction by site-directed mutagenesis. The catalytic activity has been nearly completely abolished after mutation of each of the cysteines to serine. A decrease in fluorescence on reduction by sulfide as observed for the wild-type enzyme has not been observed for any of the mutated enzymes. Mutation of a conserved valine residue to aspartate within the third flavin-binding domain led to a drastically reduced substrate affinity, for both sulfide and quinone. Two conserved histidine residues have been mutated individually to alanine. Both of the resulting enzymes exhibited a shift in the pH dependence of the SQR reaction. Polysulfide has been identified as a primary reaction product using spectroscopic and chromatographic methods. On the basis of these data, reaction mechanisms for sulfide-dependent reduction and quinone-dependent oxidation of the enzyme and for the formation of polysulfide are proposed.