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The gene for an extremely thermostable oligo-1,6-glucosidase (dextrin-6-a-D-glucanohydrolase; EC 3.2.1.10) of obligately thermophilic Bacillus thermoglucosidasius KP1006 was cloned within a 4.2-kilobase HindIII-PvuII fragment of DNA by using the plasmid pUC19 as a vector and Escherichia coli C600 as a host. The gene was transcribed, presumably from its own promoter, in E. coli. E. coli with the hybrid plasmid accumulated oligo-1,6-glucosidase mainly in the cytoplasm. The level of enzyme production was comparable to that observed for B. thermoglucosidasius. The enzyme coincided absolutely with the B. thermoglucosidasius enzyme in its molecular weight (60,000), in its electrophoretic behavior on denaturing and nondenaturing polyacrylamide gels, in the temperature dependency of its stability and activity, and in its antigenic determinants.Five p-nitrophenyl-a-D-glucopyranoside-hydrolyzing oligo-1,6-glucosidases (dextrin 6-a-D-glucanohydrolase; EC 3.2.1.10) from various Bacillus species with different ranges of growth temperature were compared for their thermostability, amino acid composition, and structural parameters calculated from amino acid composition (19). From this analysis, in conjunction with the strong site specificity of proline residues for the n-turn (2,3,8), it has been proposed that enhanced thermostability of Bacillus oligo-1,6-glucosidases would be gained by increasing the frequency of proline occurrence at n-turns and the total hydrophobic residues (19). This appeared to be given strong support by Matthews et al. (10); very recently, these authors increased the thermostability of bacteriophage T4 lysozyme by replacing alanine with proline at one of the n-turns to decrease the backbone entropy of unfolding. Site-directed mutagenesis of individual Bacillus oligo-1,6-glucosidases, followed by analysis of their altered thermostability, would provide the concrete information needed to confirm the above hypothesis. In the present study, as a first step toward fulfilling this purpose, a Bacillus thermoglucosidasius KP1006 (DSM2542; an obligate thermophile) gene coding for an extremely thermostable oligo-1,6-glucosidase was cloned and expressed in Escherichia coli.E. coli C600 (F-thi-i thr-J leuB6 lacYl tonA21 supE44 A-) and E. coli JM109 [recAl endAl gyrA96 thi hsdRI7 supE44 relAl X-A(lac-proAB) F'(traD36 proA+B+ lacIq lacZAM15)] were used (1). Aerobic cultivation was carried out at 370C in L broth (pH 7.2), consisting of 1% peptone, 0.5% yeast extract, 0.5% NaCl, 0.1% glucose, and distilled water. All DNA manipulations were as described by Maniatis et al. (9). Restriction endonucleases and T4 DNA ligase were used as specified by the supplier (Toyobo, Osaka, Japan, or Takara Shuzo, Kyoto, Japan). Cellular and extracellular oligo-1,6-glucosidase activities were assayed with p-nitrophenyl-a-D-glucopyranoside as the substrate (16,22 phenyl-cX-D-glucopyranosidase active at 60'C nor a protein cross-reactive with rabbit antiserum against B. thermoglucosidasius KP1006 oligo-1,6-glucosidase on double immunodiffu...

Extracellular-superoxide dismutase (genetic name SOD3) is a secreted anti-oxidative enzyme, and its presence in vascular walls may play an important role in protecting the vascular system against oxidative stress. Oxidative stress has been implicated in the pathogenesis of diabetic retinopathy; therefore, increases in extracellular-superoxide dismutase have been suggested to inhibit the progression of diabetic retinopathy. Incretin-based drugs such as glucagon-like peptide-1 receptor agonists are used in the treatment of type 2 diabetes. Glucagon-like peptide-1 receptor agonists are expected to function as extrapancreatic agents because the glucagon-like peptide-1 receptor is expressed not only in pancreatic tissues, but also in many other tissue types. We herein demonstrated that exendin-4, a glucagon-like peptide-1 receptor agonist, induced the expression of extracellular-superoxide dismutase in human retinal microvascular endothelial cells through epigenetic regulation. The results of the present study demonstrated that exendin-4 induced the expression of extracellular-superoxide dismutase through histone H3 acetylation at the SOD3 proximal promoter region. Moreover, plasma extracellular-superoxide dismutase concentrations in diabetic patients were elevated by incretin-based therapies. Therefore, incretin-based therapies may exert direct extrapancreatic effects in order to protect blood vessels by enhancing anti-oxidative activity.

T. Nitric oxide activates leak K ϩ currents in the presumed cholinergic neuron of basal forebrain. J Neurophysiol 98: [3397][3398][3399][3400][3401][3402][3403][3404][3405][3406][3407][3408][3409][3410] 2007. First published October 10, 2007; doi:10.1152/jn.00536.2007. Learning and memory are critically dependent on basal forebrain cholinergic (BFC) neuron excitability, which is modulated profoundly by leak K ϩ channels. Many neuromodulators closing leak K ϩ channels have been reported, whereas their endogenous opener remained unknown. We here demonstrate that nitric oxide (NO) can be the endogenous opener of leak K ϩ channels in the presumed BFC neurons. Bath application of 1 mM S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, induced a long-lasting hyperpolarization, which was often interrupted by a transient depolarization. Soluble guanylyl cyclase inhibitors prevented SNAP from inducing hyperpolarization but allowed SNAP to cause depolarization, whereas bath application of 0.2 mM 8-bromoguanosine-3Ј,5Ј-cyclomonophosphate (8-Br-cGMP) induced a similar long-lasting hyperpolarization alone. These observations indicate that the SNAP-induced hyperpolarization and depolarization are mediated by the cGMP-dependent and -independent processes, respectively. When examined with the ramp command pulse applied at -70 mV under the voltage-clamp condition, 8-Br-cGMP application induced the outward current that reversed at K ϩ equilibrium potential (E K ) and displayed Goldman-Hodgkin-Katz rectification, indicating the involvement of voltage-independent K ϩ current. By contrast, SNAP application in the presumed BFC neurons either dialyzed with the GTP-free internal solution or in the presence of 10 M Rp-8-bromo-␤-phenyl-1,N 2 -ethenoguanosine 3Ј,5Ј-cyclic monophosphorothioate sodium salt, a protein kinase G (PKG) inhibitor, induced the inward current that reversed at potentials much more negative than E K and close to the reversal potential of Na ϩ -K ϩ pump current. These observations strongly suggest that NO activates leak K ϩ channels through cGMP-PKG-dependent pathway to markedly decrease the excitability in BFC neurons, while NO simultaneously causes depolarization by the inhibition of Na ϩ -K ϩ pump through ATP depletion.

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