Structure of a signaling-competent reelin fragment revealed by X-ray crystallography and electron tomography

The large extracellular glycoprotein reelin directs neuronal migration during brain development and plays a fundamental role in layer formation. It is composed of eight tandem repeats of an approximately 380-residue unit, termed the reelin repeat, which has a central epidermal growth factor (EGF) module flanked by two homologous subrepeats with no obvious sequence similarity to proteins of known structure. The 2.05 A crystal structure of the mouse reelin repeat 3 reveals that the subrepeat assumes a beta-jelly-roll fold with unexpected structural similarity to carbohydrate-binding domains. Despite the interruption by the EGF module, the two subdomains make direct contact, resulting in a compact overall structure. Electron micrographs of a four-domain fragment encompassing repeats 3-6, which is capable of inducing Disabled-1 phosphorylation in neurons, show a rod-like shape. Furthermore, a three-dimensional molecular envelope of the fragment obtained by single-particle tomography can be fitted with four concatenated repeat 3 atomic structures, providing the first glimpse of the structural unit for this important signaling molecule.     

A diphtheria toxin receptor deficient in epidermal growth factor-like biological activity

Targeted cell ablation in animals is a powerful method for analyzing the physiological function of cell populations and generating various animal models of organ dysfunction. To achieve more specific and conditional ablation of target cells, we have developed a method termed Toxin Receptor mediated Cell Knockout (TRECK). A potential shortcoming of this method, however, is that overexpression of human heparin-binding epidermal growth factor-like growth factor (hHB-EGF) as a diphtheria toxin (DT) receptor in target cells or tissues may cause abnormalities in transgenic mice, since hHB-EGF is a member of the EGF growth factor family. To create novel DT receptors that are defective in growth factor activity and resistant to metalloprotease-cleavage, we mutated five amino acids in the extracellular EGF-like domain of hHB-EGF, which contains both DT-binding and protease-cleavage sites. Two of the resultant hHB-EGF mutants, I117A/L148V and I117V/L148V, possessed little growth factor activity but retained DT receptor activity. Furthermore, these mutants were resistant to metalloprotease-cleavage by 12-O-tetradecanoylphorbol-13-acetate stimulation, which is expected to enhance DT receptor activity. These novel DT receptors should be useful for the generation of transgenic mice by TRECK.     

Expression of ADP-ribosylation factor-like protein 8B mRNA in the brain is down-regulated in mice fed a high-fat diet

A differential display was performed to analyze differential gene expression in the brains of mice in association with dietary high beef-tallow. Consumption of a high beef-tallow diet downregulated the expression of ADP-ribosylation factor-like protein 8B (Arl8B) mRNA in the brain. Arl8B mRNA was widely expressed in the mouse brain, including primary neuronal cells. The current study indicates that green fluorescent protein-fused Arl8B protein accumulated at the growth cones in primary neuronal cells, and that protrusions of human embryonic kidney 293 (HEK293) cells were significantly elongated by overexpression of Arl8B, suggesting an important role of Arl8B in neurite formation.     

The Developmentally Regulated Osteoblast Phosphodiesterase GDE3 Is Glycerophosphoinositol-specific and Modulates Cell Growth

The glycerophosphodiester phosphodiesterase enzyme family involved in the hydrolysis of glycerophosphodiesters has been characterized in bacteria and recently identified in mammals. Here, we have characterized the activity and function of GDE3, one of the seven mammalian enzymes. GDE3 is up-regulated during osteoblast differentiation and can affect cell morphology. We show that GDE3 is a glycerophosphoinositol (GroPIns) phosphodiesterase that hydrolyzes GroPIns, producing inositol 1-phosphate and glycerol, and thus suggesting specific roles for this enzyme in GroPIns metabolism. Substrate specificity analyses show that wild-type GDE3 selectively hydrolyzes GroPIns over glycerophosphocholine, glycerophosphoethanolamine, and glycerophosphoserine. A single point mutation in the catalytic domain of GDE3 (GDE3R231A) leads to loss of GroPIns enzymatic hydrolysis, identifying an arginine residue crucial for GDE3 activity. After heterologous GDE3 expression in HEK293T cells, phosphodiesterase activity is detected in the extracellular medium, with no effect on the intracellular GroPIns pool. Together with the millimolar concentrations of calcium required for GDE3 activity, this predicts an enzyme topology with an extracellular catalytic domain. Interestingly, GDE3 ectocellular activity is detected in a stable clone from a murine osteoblast cell line, further confirming the activity of GDE3 in a more physiological context. Finally, overexpression of wild-type GDE3 in osteoblasts promotes disassembly of actin stress fibers, decrease in growth rate, and increase in alkaline phosphatase activity and calcium content, indicating a role for GDE3 in induction of differentiation. Thus, we have identified the GDE3 substrate GroPIns as a candidate mediator for osteoblast proliferation, in line with the GroPIns activity observed previously in epithelial cells.The glycerophosphodiester phosphodiesterases (GP-PDEs)⁵ were initially characterized in bacteria, where they have functional roles for production of metabolic carbon and phosphate sources from glycerophosphodiesters (1, 2) and in adherence to and degradation of mammalian host-cell membranes (3). The GP-PDEs have a catalytic region of 56 amino acids (4). After their characterization in bacteria, mammalian glycerophosphodiesterases were identified, with the definition of a family of seven members (5). The first of these, GDE1, is an interactor of regulator of G-protein signaling (RGS)16, and was subsequently defined as a GP-PDE regulated by G-protein signaling (4). Indeed, GDE1 expression in HEK293T cells showed increased enzymatic activity upon α/β-adrenergic and lysophospholipid receptor stimulation (4). The second member, GDE2, was isolated by homology searches in neuronal tissues and its physiological role involves neuronal differentiation (6, 7). In contrast, GDE3 has been characterized as a marker of osteoblast differentiation and was isolated through a differential display method (8). GDE4 was isolated only recently with three-dimensional modeling defining it as a GP-PDE, although no functional activity has been correlated to its expression (9). The remaining members were cloned following data base searches, with further studies required for the definition of their properties (5). The diversity among these family members, in terms of tissue distribution, subcellular localization, and substrate specificity, suggests they selectively regulate biological functions and have distinct physiological roles (5).The only GP-PDE activity that has been biochemically characterized to date followed GDE1 overexpression in HEK293T cells, which showed a selectivity for the glycerophosphoinositols (GPIs) as substrate (4), in contrast to the bacterial GP-PDEs that show broad substrate specificities with respect to the alcohol moiety of the glycerophosphodiesterases (1, 2). The GPIs are naturally occurring, biologically active metabolites of the phosphoinositides that were originally investigated in the context of Ras-transformed cells (10). They are present in virtually all cell types, where their intracellular levels can also be modulated according to cell activation, differentiation, and development (Refs. 11 and 12 and references therein). Recently, glycerophosphoinositol (GroPIns) was characterized as a mediator of purinergic and adrenergic regulation of PCCl₃ thyroid cell proliferation (13), while GroPIns 4-phosphate (GroPIns4P) has been shown to induce reorganization of the actin cytoskeleton in fibroblasts and in T-lymphocytes, by promoting a sustained and robust activation of the Rho GTPases (14–16).The GPIs appear to rapidly equilibrate across the plasma membrane when added exogenously to cells, to exert their actions within the cell (12). The plasma membrane transporter for GroPIns characterized in yeast is the protein GIT1 (17), with one of its orthologs in mammalian cells identified as the human permease Glut2 (18). This specific transporter has been proposed to mediate both GroPIns uptake and release, which depends on the GroPIns concentration gradient across the plasma membrane. Under physiological conditions, this gradient can arise from the formation of GPIs from the phosphoinositides inside cells following activation of a specific isoform of phospholipase A₂, PLA₂IVα (13, 19).The release of the GPIs into the extracellular medium can affect their paracrine targets (16) or initiate their catabolism. This is supported by our characterization of GDE1 activity, and now of GDE3 activity, both of which show a substrate selectivity toward GroPIns, and catalytic activity after heterologous expression that can only be monitored in the extracellular space. Interestingly, GDE3 activity appears to be related to modulation of osteoblast functions, delineating a role for GDE3 in promoting osteoblast differentiation, and mainly regulating osteoblast proliferation.     

Inhibitory effects of coumarin and acetylene constituents from the roots of Angelica furcijuga on D-galactosamine/lipopolysaccharide-induced liver injury in mice and on nitric oxide production in lipopolysaccharide-activated mouse peritoneal macrophages

The methanolic extract (200 mg/kg, p.o. and i.p.), principal coumarin constituents (isoepoxypteryxin, anomalin, and praeroside IV), and a polyacetylene constituent (falcarindiol) (25 mg/kg, i.p.) from the roots of Angelica furcijuga protected the liver injury induced by D-galactosamine (D-GalN)/lipopolysaccharide (LPS) in mice. In in vitro experiments, coumarin constituents (hyuganins A-D, anomalin, pteryxin, isopteryxin, and suksdorfin) and polyacetylene constituents [(-)-falcarinol and falcarindiol] substantially inhibited LPS-induced NO and/or TNF-alpha production in mouse peritoneal macrophages, and isoepoxypteryxin inhibited D-GalN-induced cytotoxicity in primary cultured rat hepatocytes. Furthermore, hyuganin A, anomalin, and isopteryxin inhibited the decrease in cell viability by TNF-alpha in L929 cells.     

The throat flora and its mitogenic activity in patients with Kawasaki disease

The etiology of Kawasaki disease (KD) remains unknown, although some infectious organism has been suggested as the cause. Recent studies suggest that some bacterial toxins with superantigen activity are involved in its pathogenesis, but no specific bacterial toxin has yet been identified. Throat swabs for bacterial culture were obtained from 21 patients with KD and 20 with other febrile illnesses as controls. Mitogenic activity in culture supernatants obtained from individual bacterial strains was measured by lymphocyte proliferation assay. Sixty-one bacterial strains were isolated from KD patients, and 62 strains from control patients. There was no apparent difference in bacterial species in the throat flora between KD patients and febrile controls. Moreover, total and individual mitogenic activity of strains from KD patients was no greater than that of strains from febrile controls. The bacterial superantigen activity of throat flora may not play a major role in the pathogenesis of KD.     

Adenosine A2A receptor facilitates calcium-dependent protein secretion through the activation of protein kinase A and phosphatidylinositol-3 kinase in PC12 cells

Adenosine modulates a variety of cellular functions including calcium-dependent exocytosis. Activation of adenosine A(2A) receptor (A(2A)-R) facilitates neurotransmitter release in some cell types, although the underlying mechanisms are not fully understood. In this study, we found that treatment of PC12 cells with the A(2A)-R agonist CGS21680 promotes calcium-evoked secretion of the fusion protein between neuropeptide Y and modified yellow fluorescence protein (NPY-Venus). CGS21680 treatment of PC12 cells transiently increased the phosphorylation of p38 and JNK MAP kinases and Akt, as well as that of ATF2 and CREB, reaching maximal levels at around 10-15 min of CGS21680 treatment. Importantly, pretreatment of PC12 cells with the PI3K inhibitor LY294002, together with the protein kinase A (PKA) inhibitor KT5720, significantly inhibited CGS21680 enhancement of calcium-dependent NPY-Venus release. Moreover, expression of a dominant-negative form of Akt and the PKA inhibitory polypeptide protein kinase inhibitor (PKI) co-operatively inhibited the facilitating effect of CGS21680 on secretion of NPY-Venus. These data suggest that the PI3K-Akt and PKA pathways play a critical role in A(2A)-R-mediated facilitation of calcium-dependent secretion. We also found that CGS21680 treatment promoted recruitment of the NPY-Venus-containing vesicles to the proximity of the plasma membrane at around 10-15 min of CGS21680 treatment, which may in part account for the facilitated secretion by A(2A)-R activation.