Structure and solution properties of enzymatically synthesized glycogen

Recently, a new enzymatic process for glycogen production was developed. In this process, short-chain amylose is used as a substrate for branching enzymes (BE, EC 2.4.1.18). The molecular weight of the enzymatically synthesized glycogen (ESG) depends on the size and concentration of the substrate. Structural and physicochemical properties of ESG were compared to those of natural source glycogen (NSG). The average chain length, interior chain length, and exterior chain length of ESG were 8.2-11.6, 2.0-3.3, and 4.2-7.6, respectively. These values were within the range of variation of NSG. The appearances of both ESG and NSG in solution were opalescent (milky white and slightly bluish). Furthermore, transmission electron microscopy and atomic force microscopy showed that ESG molecules formed spherical particles, and that there were no differences between ESG and NSG. Viscometric analyses also showed the spherical nature of both glycogens. When ESG and NSG were treated with pullulanase, a glucan-hydrolyzing enzyme known to degrade glycogen only on its surface portion, both glycogens were similarly degraded. These analyses revealed that ESG shares similar molecular shapes and surface properties with NSG.     

eNOS-β-Actin Interaction Contributes to Increased Peroxynitrite Formation during Hyperoxia in Pulmonary Artery Endothelial Cells and Mouse Lungs

Oxygen toxicity is the most severe side effect of oxygen therapy in neonates and adults. Pulmonary damage of oxygen toxicity is related to the overproduction of reactive oxygen species (ROS). In the present study, we investigated the effect of hyperoxia on the production of peroxynitrite in pulmonary artery endothelial cells (PAEC) and mouse lungs. Incubation of PAEC under hyperoxia (95% O₂) for 24 h resulted in an increase in peroxynitrite formation. Uric acid, a peroxynitrite scavenger, prevented hyperoxia-induced increase in peroxynitrite. The increase in peroxynitrite formation is accompanied by increases in nitric oxide (NO) release and endothelial NO synthase (eNOS) activity. We have previously reported that association of eNOS with β-actin increases eNOS activity and NO production in lung endothelial cells. To study whether eNOS-β-actin association contributes to increased peroxynitrite production, eNOS-β-actin interaction were inhibited by reducing β-actin availability or by using a synthetic peptide (P326TAT) containing a sequence corresponding to the actin binding site on eNOS. We found that disruption of eNOS-β-actin interaction prevented hyperoxia-induced increases in eNOS-β-actin association, eNOS activity, NO and peroxynitrite production, and protein tyrosine nitration. Hyperoxia failed to induce the increases in eNOS activity, NO and peroxynitrite formation in COS-7 cells transfected with plasmids containing eNOS mutant cDNA in which amino acids leucine and tryptophan were replaced with alanine in the actin binding site on eNOS. Exposure of mice to hyperoxia resulted in significant increases in eNOS-β-actin association, eNOS activity, and protein tyrosine nitration in the lungs. Our data indicate that increased association of eNOS with β-actin in PAEC contributes to hyperoxia-induced increase in the production of peroxynitrite which may cause nitrosative stress in pulmonary vasculature.     

Nitric Oxide Mediates the Glutamate-dependent Pathway for Neurotransmission in Sepia officinalis Chromatophore Organs

Chromatophore organs are complex and unique structures responsible for the variety of body coloration patterns used by cephalopods to communicate and camouflage. They are formed by a pigment-containing cytoelastic sacculus, surrounded by muscle fibers directly innervated from the brain. Muscle contraction and relaxation are responsible for expansion and retraction of the pigment-containing cell. Their functioning depends on glutamate and Phe-Met-Arg-Phe-NH₂-related peptides, which induce fast and slow cell expansion, respectively, and 5-hydroxytryptamine, which induces retraction. Apart from these three substances and acetylcholine, which acts presynaptically, no other neuroactive compounds have so far been found to be involved in the neuroregulation of chromatophore physiology, and the detailed signaling mechanisms are still little understood. Herein, we disclose the role of nitric oxide (NO) as mediator in one of the signaling pathways by which glutamate activates body patterning. NO and nitric-oxide synthase have been detected in pigment and muscle fibers of embryo, juvenile, and adult chromatophore organs from Sepia officinalis. NO-mediated Sepia chromatophore expansion operates at slower rate than glutamate and involves cGMP, cyclic ADP-ribose, and ryanodine receptor activation. These results demonstrate for the first time that NO is an important messenger in the long term maintenance of the body coloration patterns in Sepia.     

Regulation of AMPA Receptor Trafficking and Function by Glycogen Synthase Kinase 3

Accumulating evidence suggests that glycogen synthase kinase 3 (GSK-3) is a multifunctional kinase implicated in neuronal development, mood stabilization, and neurodegeneration. However, the synaptic actions of GSK-3 are largely unknown. In this study, we examined the impact of GSK-3 on AMPA receptor (AMPAR) channels, the major mediator of excitatory transmission, in cortical neurons. Application of GSK-3 inhibitors or knockdown of GSK-3 caused a significant reduction of the amplitude of miniature excitatory postsynaptic current (mEPSC), a readout of the unitary strength of synaptic AMPARs. Treatment with GSK-3 inhibitors also decreased surface and synaptic GluR1 clusters on dendrites and increased internalized GluR1 in cortical cultures. Rab5, the small GTPase controlling the transport from plasma membrane to early endosomes, was activated by GSK-3 inhibitors. Knockdown of Rab5 prevented GSK-3 inhibitors from regulating mEPSC amplitude. Guanyl nucleotide dissociation inhibitor (GDI), which regulates the cycle of Rab5 between membrane and cytosol, formed an increased complex with Rab5 after treatment with GSK-3 inhibitors. Blocking the function of GDI occluded the effect of GSK-3 inhibitors on mEPSC amplitude. In cells transfected with the non-phosphorylatable GDI mutant, GDI(S45A), GSK-3 inhibitors lost the capability to regulate GDI-Rab5 complex, mEPSC amplitude, and AMPAR surface expression. These results suggest that GSK-3, via altering the GDI-Rab5 complex, regulates Rab5-mediated endocytosis of AMPARs. It provides a potential mechanism underlying the role of GSK-3 in synaptic transmission and plasticity.     

Suppression of Adipocyte Differentiation by Aldo-keto Reductase 1B3 Acting as Prostaglandin F2�� Synthase

Prostaglandin (PG) F_2α suppresses adipocyte differentiation by inhibiting the function of peroxisome proliferator-activated receptor γ. However, PGF_2α synthase (PGFS) in adipocytes remains to be identified. Here, we studied the expression of members of the aldo-keto reductase (AKR) 1B family acting as PGFS during adipogenesis of mouse 3T3-L1 cells. AKR1B3 mRNA was expressed in preadipocytes, and its level increased about 4-fold at day 1 after initiation of adipocyte differentiation, and then quickly decreased the following day to a level lower than that in the preadipocytes. In contrast, the mRNA levels of Akr1b8 and 1b10 were clearly lower than that level of Akr1b3 in preadipocytes and remained unchanged during adipogenesis. The transient increase in Akr1b3 during adipogenesis was also observed by Western blot analysis. The mRNA for the FP receptor, which is selective for PGF_2α, was also expressed in preadipocytes. Its level increased about 2-fold within 1 h after the initiation of adipocyte differentiation and was maintained at almost the same level throughout adipocyte differentiation. The small interfering RNA for Akr1b3, but not for Akr1b8 or 1b10, suppressed PGF_2α production and enhanced the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ, fatty acid-binding protein 4 (aP2), and stearoyl-CoA desaturase. Moreover, an FP receptor agonist, Fluprostenol, suppressed the expression of those adipogenic genes in 3T3-L1 cells; whereas an FP receptor antagonist, AL-8810, efficiently inhibited the suppression of adipogenesis caused by the endogenous PGF_2α. These results indicate that AKR1B3 acts as the PGFS in adipocytes and that AKR1B3-produced PGF_2α suppressed adipocyte differentiation by acting through FP receptors.     

Phosphorylation of mCRY2 at Ser557 in the Hypothalamic Suprachiasmatic Nucleus of the Mouse

Cryptochrome1 and 2 play a critical role in the molecular oscillations of the circadian clocks of central and peripheral tissues in mammals. Mouse Cryptochrome2 (mCRY2) is phosphorylated at Ser557 in the liver, in which the Ser557-phosphorylated form accumulates during the night in parallel with mCRY2 protein. Phosphorylation of mCRY2 at Ser557 allows subsequent phosphorylation at Ser553 by glycogen synthase kinase-3β (GSK-3β), resulting in efficient degradation of mCRY2 by a proteasome pathway. In the present study, we found that mCRY2 is phosphorylated at Ser557 also in the region of the mouse brain containing the suprachiasmatic nucleus (SCN), the central circadian clock tissue. Daily fluctuation of the Ser557-phosphorylation level in the SCN region suggests an important role of sequential phosphorylation of Ser557 and Ser553 in the rhythmic degradation of mCRY2 in both central and peripheral clocks of mice.     

Genome-wide expression analysis of yeast response during exposure to 4°C

Adaptation to temperature fluctuation is essential for the survival of all living organisms. Although extensive research has been done on heat and cold shock responses, there have been no reports on global responses to cold shock below 10°C or near-freezing. We examined the genome-wide expression in Saccharomyces cerevisiae, following exposure to 4°C. Hierarchical cluster analysis showed that the gene expression profile following 4°C exposure from 6 to 48 h was different from that at continuous 4°C culture. Under 4°C exposure, the genes involved in trehalose and glycogen synthesis were induced, suggesting that biosynthesis and accumulation of those reserve carbohydrates might be necessary for cold tolerance and energy preservation. The observed increased expression of phospholipids, mannoproteins, and cold shock proteins (e.g., TIP1) is consistent with membrane maintenance and increased permeability of the cell wall at 4°C. The induction of heat shock proteins and glutathione at 4°C may be required for revitalization of enzyme activity, and for detoxification of active oxygen species, respectively. The genes with these functions may provide the ability of cold tolerance and adaptation to yeast cells.     

Covalent Binding of 1,N 6-ethenoadenosine diphosphate to catalytic and noncatalytic sites of chloroplast ATP synthase

Catalytic and noncatalytic sites of the chloroplast coupling factor (CF₁) were selectively modified by incubation with the dialdehyde derivative of fluorescent adenosine diphosphate analog 1,N⁶-ethenoadenosine diphosphate. The modified CF₁ was reconstituted with EDTA-treated thylakoid membranes of chloroplasts. The effects of light-induced transmembrane proton gradient and phosphate ions on the fluorescence of 1,N⁶-ethenoadenosine diphosphate, covalently bound to the catalytic sites of ATP synthase, were studied. Quenching of fluorescence of covalently bound 1,N⁶-ethenoadenosine diphosphate was observed under illumination of thylakoid membranes with saturating white light. Addition of inorganic phosphate to the reaction mixture in the dark increased the fluorescence of the label. Quenching reappeared under repeated illumination; however, addition of phosphate ions had no effect on the fluorescence yield in this case. When 1,N⁶-ethenoadenosine diphosphate was covalently bound to noncatalytic sites of ATP synthase, no similar fluorescence changes were observed. The relation between the observed changes of 1,N⁶-ethenoadenosine diphosphate fluorescence and the mechanism of energy-dependent structural changes in the catalytic site of ATP synthase is discussed.     

Interaction between glycogenin and glycogen synthase

Glycogen synthase plays a key role in regulating glycogen metabolism. In a search for regulators of glycogen synthase, a yeast two-hybrid study was performed. Two glycogen synthase-interacting proteins were identified in human skeletal muscle, glycogenin-1, and nebulin. The interaction with glycogenin was found to be mediated by the region of glycogenin which contains the 33 COOH-terminal amino acid residues. The regions in glycogen synthase containing both NH2- and COOH-terminal phosphorylation sites are not involved in the interaction. The core segment of glycogen synthase from Glu21 to Gly503 does not bind COOH-terminal fragment of glycogenin. However, this region of glycogen synthase binds full-length glycogenin indicating that glycogenin contains at least one additional interacting site for glycogen synthase besides the COOH-terminus. We demonstrate that the COOH-terminal fragment of glycogenin can be used as an effective high affinity reagent for the purification of glycogen synthase from skeletal muscle and liver.