Chemo-enzymatic synthesis of eel calcitonin glycosylated at two sites with the same and different carbohydrate structures

Naturally occurring glycopeptides and glycoproteins usually contain more than one glycosylation site, and the structure of the carbohydrate attached is often different from site to site. Therefore, synthetic methods for preparing peptides and proteins that are glycosylated at multiple sites, possibly with different carbohydrate structures, are needed. Here, we report a chemo-enzymatic approach for accomplishing this. Complex-type oligosaccharides were introduced to the calcitonin derivatives that contained two N-acetyl-D-glucosamine (GlcNAc) residues at different sites by treatment with Mucor hiemalis endo-beta-N-acetylglucosaminidase. Using this enzymatic transglycosylation reaction, three glycopeptides were produced, a calcitonin derivative with the same complex-type carbohydrate at two sites, and two calcitonin derivatives each with one complex-type carbohydrate and one GlcNAc. Starting from the derivatives with one complex-type carbohydrate and one GlcNAc, a high-mannose-type oligosaccharide was successfully transferred to the remaining GlcNAc using another endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae. Thus, we were able to obtain glycopeptides containing not only two complex-type carbohydrates, but also both complex and high-mannose-type oligosaccharides in a single molecule. Using the resultant glycosylated calcitonin derivatives, the effects of di-N-glycosylation on the structure and the activity of calcitonin were studied. The effect appeared to be predictable from the results of mono-N-glycosylated calcitonin derivatives.     

Pioglitazone ameliorates insulin resistance and diabetes by both adiponectin-dependent and -independent pathways

Thiazolidinediones have been shown to up-regulate adiponectin expression in white adipose tissue and plasma adiponectin levels, and these up-regulations have been proposed to be a major mechanism of the thiazolidinedione-induced amelioration of insulin resistance linked to obesity. To test this hypothesis, we generated adiponectin knock-out (adipo-/-) ob/ob mice with a C57B/6 background. After 14 days of 10 mg/kg pioglitazone, the insulin resistance and diabetes of ob/ob mice were significantly improved in association with significant up-regulation of serum adiponectin levels. Amelioration of insulin resistance in ob/ob mice was attributed to decreased glucose production and increased AMP-activated protein kinase in the liver but not to increased glucose uptake in skeletal muscle. In contrast, insulin resistance and diabetes were not improved in adipo-/-ob/ob mice. After 14 days of 30 mg/kg pioglitazone, insulin resistance and diabetes of ob/ob mice were again significantly ameliorated, which was attributed not only to decreased glucose production in the liver but also to increased glucose uptake in skeletal muscle. Interestingly, adipo-/-ob/ob mice also displayed significant amelioration of insulin resistance and diabetes, which was attributed to increased glucose uptake in skeletal muscle but not to decreased glucose production in the liver. The serum-free fatty acid and triglyceride levels as well as adipocyte sizes in ob/ob and adipo-/-ob/ob mice were unchanged after 10 mg/kg pioglitazone but were significantly reduced to a similar degree after 30 mg/kg pioglitazone. Moreover, the expressions of TNFalpha and resistin in adipose tissues of ob/ob and adipo-/-ob/ob mice were unchanged after 10 mg/kg pioglitazone but were decreased after 30 mg/kg pioglitazone. Thus, pioglitazone-induced amelioration of insulin resistance and diabetes may occur adiponectin dependently in the liver and adiponectin independently in skeletal muscle.     

Viscoelastic and dynamic nonlinear properties of airway smooth muscle tissue: roles of mechanical force and the cytoskeleton

The viscoelastic and dynamic nonlinear properties of guinea pig tracheal smooth muscle tissues were investigated by measuring the storage (G') and loss (G") moduli using pseudorandom small-amplitude length oscillations between 0.12 and 3.5 Hz superimposed on static strains of either 10 or 20% of initial length. The G" and G' spectra were interpreted using a linear viscoelastic model incorporating damping (G) and stiffness (H), respectively. Both G and H were elevated following an increase in strain from 10 to 20%. There was no change in harmonic distortion (K(d)), an index of dynamic nonlinearity, between 10 and 20% strains. Application of methacholine at 10% strain significantly increased G and H while it decreased K(d). Cytochalasin D, isoproterenol, and HA-1077, a Rho-kinase inhibitor, significantly decreased both G and H but increased K(d). Following cytochalasin D, G, H, and K(d) were all elevated when mean strain increased from 10 to 20%. There were no changes in hysteresivity, G/H, under any condition. We conclude that not all aspects of the viscoelastic properties of tracheal smooth muscle strips are similar to those previously observed in cultured cells. We attribute these differences to the contribution of the extracellular matrix. Additionally, using a network model, we show that the dynamic nonlinear behavior, which has not been observed in cell culture, is associated with the state of the contractile stress and may derive from active polymerization within the cytoskeleton.     

Control of axonal sprouting and dendrite branching by the Nrg-Ank complex at the neuron-glia interface

Neurons are highly polarized cells with distinct subcellular compartments, including dendritic arbors and an axon. The proper function of the nervous system relies not only on correct targeting of axons, but also on development of neuronal-class-specific geometry of dendritic arbors [1-4]. To study the intercellular control of the shaping of dendritic trees in vivo, we searched for cell-surface proteins expressed by Drosophila dendritic arborization (da) neurons [5-7]. One of them was Neuroglian (Nrg), a member of the Ig superfamily ; Nrg and vertebrate L1-family molecules have been implicated in various aspects of neuronal wiring, such as axon guidance, axonal myelination, and synapse formation [9-12]. A subset of the da neurons in nrg mutant embryos exhibited deformed dendritic arbors and abnormal axonal sprouting. Our functional analysis in a cell-type-selective manner strongly suggested that those da neurons employed Nrg to interact with the peripheral glia for suppressing axonal sprouting and for forming second-order dendritic branches. At least for the former role, Nrg functioned in concert with the intracellular adaptor protein Ankyrin (Ank) [13]. Thus, the neuron-glia interaction that is mediated by Nrg, together with Ank under some situations, contributes to axonal and dendritic morphogenesis.     

Purification of glycogen debranching enzyme from porcine brain: evidence for glycogen catabolism in the brain

Amylo-1,6-glucosidase from porcine brain was purified to homogeneity by ammonium sulfate fractionation, followed by sequential steps of liquid chromatography on DEAE-Sephacel, Sephacryl S-300, and Super Q. The purified enzyme had both maltooligosaccharide transferase and amylo-1,6-glucosidase activities within a single polypeptide chain, and the combination of these two activities removed the branches of phosphorylase limit dextrin. Based on these results, the purified enzyme was identified as a glycogen debranching enzyme (GDE). The molecular weight of the brain GDE was 170,000 by gel-filtration and 165,000 by reducing SDS-PAGE. The pH profile of maltooligosaccharide transferase activity coincided with that of the amylo-1,6-glucosidase activity (pH optimum at 6.0). The existence of GDE as well as glycogen phosphorylase in the brain explains brain glycogenolysis fully and supports the hypothesis that glycogen is a significant source of energy in this organ.     

An alpha-amylase homologue, aah3, encodes a GPI-anchored membrane protein required for cell wall integrity and morphogenesis in Schizosaccharomyces pombe

Glycosylphosphatidylinositol (GPI)-anchored proteins are essential for normal cellular morphogenesis and have an additional role in mediating cross-linking of glycoproteins to cell wall glucan in yeast cells. Although many GPI-anchored proteins have been characterized in Saccharomyces cerevisiae, none have been reported for well-characterized GPI-anchored proteins in Schizosaccharomyces pombe to date. Among the putative GPI-anchored proteins in S. pombe, four alpha-amylase homologs (Aah1p-Aah4p) have putative signal sequences and C-terminal GPI anchor addition signals. Disruption of aah3(+) resulted in a morphological defect and hypersensitivity to cell wall-degrading enzymes. Biochemical analysis showed that Aah3p is an N-glycosylated, GPI-anchored membrane protein localized in the membrane and cell wall fractions. Conjugation and sporulation were not affected by the aah3(+) deletion, but the ascal wall of aah3Delta cells was easily lysed by hydrolases. Expression of aah3 alleles in which the conserved aspartic acid and glutamic acid residues required for hydrolase activity were replaced with alanine residues failed to rescue the morphological and ascal wall defects of aah3Delta cells. Taken together, these results indicate that Aah3p is a GPI-anchored protein and is required for cell and ascal wall integrity in S. pombe.     

Camellianoside, a novel antioxidant glycoside from the leaves of Camellia japonica

A novel flavonol glycoside named camellianoside and three known flavonol glycosides were isolated from the leaves of Camellia japonica. The structure of camellianoside was established as quercetin-3-O-beta-D-xylopyranosyl-(1-->3)-O-alpha-L-rhamnopyranosyl-(1-->6)-O-beta-D-glucopyranoside by spectroscopic and chemical methods. The antioxidant activities of these glycosides evaluated by the diphenylpicrylhydrazyl (DPPH) radical scavenging reaction was higher than those of L-cysteine and L-ascorbic acid used as the reference antioxidants.