Cloning and characterization of a sucrase from Leuconostoc mesenteroides

A sucrase gene from Leuconostoc mesenteroides was cloned and expressed in Escherichia coli. The cloned enzyme did not show dextransucrase or sucrose phosphorylase activity. HPLC and GC-MS analyses of the sucrase products indicated the presence of fructose and glucose in equimolar amounts. IPTG induction did not increase sucrase activity in E. coli indicating that the cloned gene may be transcribed from its own promoter. To our knowledge, this is the first sucrase cloned from L. mesenteroides that has invertase activity.     

Spliceosome-targeted therapies trigger an antiviral immune response in triple-negative breast cancer

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Blood pressure lability in the sympathectomized rat

Intra-aortic blood pressure (BP) was measured in conscious rats after early chronic destruction of the peripheral sympathetic nervous system (SNS) with guanethidine. In sympathectomized rats, the mean level of BP was not different from that of control rats but its variability was markedly increased. These results indicate that functional integrity of the SNS is of primary importance for the short-term control of BP but is not essential for its long-term maintenance.     

Cellular distribution of elongation factor EF1 in wheat germ]

Cellular distribution of elongation factors (EF1) from imbibed then redessicated wheat embryos is determined after purification and analytical gel electrophoresis of soluble and ribosome-bound factors. Two heavy forms (EF1 H, mol. wt, 250 000) are found in cytosol while ribosome-bound factors contain a light form (EF1L, mol. wt, 45 000) with the greatest activity and a heavy form (mol. wt, 160 000) which might well be an intermediary in the recycling of ribosomal factor EF1L to soluble factor EF1H.     

Nalpha-trinitrophenyl glucagon: an inhibitor of glucagon-stimulated cyclic AMP production and its effects on glycogenolysis.

Nalpha-Trinitrophenyl glucagon was prepared by reaction with trinitrobenzene sulfonic acid and purified by ion-exchange chromatography. This derivative has essentially no ability to activate adenylate cyclase from rat liver nor to increase the levels of cyclic AMP in isolated hepatocytes nor to stimulate protein kinase activity. This derivative also can act as a glucagon antagonist with regard to cyclic AMP production and can decrease the degree of stimulation of adenylate cyclase caused by glucagon, as well as lowering the glucagon-stimulated elevation of cyclic AMP levels in intact hepatocytes. Nevertheless, this derivative is capable of activating glycogenolysis in isolated hepatocytes and in augmenting the effect of glucagon on glycogenolysis. This metabolic effect of the glucagon derivative thus appears to occur independent of changes in cyclic AMP levels. These results suggest that glucagon can also activate glycogenolysis by a cyclic AM-independent process.     

Regulation of photoreceptor phosphodiesterase (PDE6) by phosphorylation of its inhibitory gamma subunit re-evaluated.

Phosphorylation of the inhibitory gamma subunit (Pgamma) of rod cGMP phosphodiesterase (PDE6) has been reported to turn off visual excitation without the requirement for inactivation of the photoreceptor G-protein transducin. We evaluated the significance of Pgamma phosphorylation for PDE6 regulation by preparing Pgamma stoichiometrically phosphorylated at Thr(22) or at Thr(35). Phosphorylation of Pgamma at either residue caused a minor decrease--not the previously reported increase--in the ability of Pgamma to inhibit catalysis at the active site of purified PDE6 catalytic dimers. Likewise, Pgamma phosphorylation had little effect on its potency to inhibit transducin-activated PDE6 depleted of its endogenous Pgamma subunits. The strength of Pgamma interaction with the regulatory GAF domain of PDE6 was reduced severalfold upon Pgamma phosphorylation at Thr(22) (but not Thr(35)), as judged by allosteric changes in cGMP binding to these noncatalytic sites on the enzyme (Mou, H., and Cote, R. H. (2001) J. Biol. Chem. 276, 27527-27534). In contrast, the effects of Pgamma phosphorylation on its interactions with activated transducin were much more pronounced. Phosphorylation of Pgamma at either Thr(22) or Thr(35) greatly diminished its ability to bind activated transducin, consistent with earlier work. In situ phosphorylation of Pgamma by endogenous rod outer segment kinases was enhanced severalfold upon light activation, but only approximately 10% of the endogenous Pgamma was phosphorylated. This is attributed to Pgamma being a poor substrate for protein kinases when associated with the PDE6 holoenzyme. We conclude that, contrary to previous reports, Pgamma phosphorylation at either Thr(22) or Thr(35) modestly weakens its direct interactions with PDE6. However, Pgamma phosphorylation subsequent to its dissociation from PDE6 is likely to abolish its binding to activated transducin and may serve to make phosphorylated Pgamma available to regulate other signal transduction pathways (e.g. mitogen-activated protein kinase; Wan, K. F., Sambi, B. S., Frame, M., Tate, R., and Pyne, N. J. (2001) J. Biol. Chem. 276, 37802-37808) in photoreceptor cells.