Production of α-Glucosidase and Its Role in Regulation of Amylase Production by Bacillus circulons F-2

Bacillus circulans F-2 requires a special carbon source or cultural conditions for amylase production. The α-glucosidase production of this bacterium was studied in various cultural conditions with measured glucose concèntrations. High amylase production was always accompanied by low α-glucosidase production and the absence of glucose in culture broth. Usually higher α-glucosidase production was observed in cultural conditions where little amylase was produced. In the presence of 1-deoxynojirimycin, an inhibitor of α-glucosidase activity, the bacterium produced significant amounts of amylase even in conditions giving high α-glucosidase production. It was concluded that the special requirement of this bacterium to produce amylase is effected by its high sensitivity to glucose repression and by the production of α-glucosidase which leads to the formation of glucose. Production of α-glucosidase was, like that of amylase, induced by maltooligosaccharides and repressed by glucose, but both its induction and repression are less sensitive to glucose than those of amylase.     

Deletion of the Mitochondrial Flavoprotein Apoptosis Inducing Factor (AIF) Induces β-Cell Apoptosis and Impairs β-Cell Mass

Background

Apoptosis is a hallmark of β-cell death in both type 1 and type 2 diabetes mellitus. Understanding how apoptosis contributes to β-cell turnover may lead to strategies to prevent progression of diabetes. A key mediator of apoptosis, mitochondrial function, and cell survival is apoptosis inducing factor (AIF). In the present study, we investigated the role of AIF on β-cell mass and survival using the Harlequin (Hq) mutant mice, which are hypomorphic for AIF.

Methodology/Principal Findings

Immunohistochemical evaluation of pancreata from Hq mutant mice displayed much smaller islets compared to wild-type mice (WT). Analysis of β-cell mass in these mice revealed a greater than 4-fold reduction in β-cell mass together with an 8-fold increase in β-cell apoptosis. Analysis of cell cycle dynamics, using BrdU pulse as a marker for cells in S-phase, did not detect significant differences in the frequency of β-cells in S-phase. In contrast, double staining for phosphorylated Histone H3 and insulin showed a 3-fold increase in β-cells in the G2 phase in Hq mutant mice, but no differences in M-phase compared to WT mice. This suggests that the β-cells from Hq mutant mice are arrested in the G2 phase and are unlikely to complete the cell cycle. β-cells from Hq mutant mice display increased sensitivity to hydrogen peroxide-induced apoptosis, which was confirmed in human islets in which AIF was depleted by siRNA. AIF deficiency had no effect on glucose stimulated insulin secretion, but the impaired effect of hydrogen peroxide on β-cell function was potentiated.

Conclusions/Significance

Our results indicate that AIF is essential for maintaining β-cell mass and for oxidative stress response. A decrease in the oxidative phosphorylation capacity may counteract the development of diabetes, despite its deleterious effects on β-cell survival.     

Covalent Modification of the NF-κB Essential Modulator (NEMO) by a Chemical Compound Can Regulate Its Ubiquitin Binding Properties in Vitro

Post-translational modification by ubiquitin plays important roles in multiple physiological and pathological processes. Ubiquitin-binding proteins play a critical role in recognizing and relaying polyubiquitin-based signaling. NEMO (NF-κB Essential Modulator) is a central player in canonical NF-κB signaling whose major function is to bind to Lys-63- and/or M1- (or linear) linked polyubiquitin chains generated in response to cell stimulation. Here we show that Withaferin A (WA), a steroidal lactone, causes a change in NEMO''s interaction with specific types of polyubiquitin chains in vitro. WA induces full-length recombinant NEMO to bind to long Lys-48-linked polyubiquitin chains but not tetra-ubiquitin species. Significantly, the UBAN (ubiquitin binding in ABIN and NEMO) domain, essential for the ability of NEMO to bind M1/Lys-63-linked polyubiquitin, is dispensable for the WA-induced gain-of-function activity. Mass spectrometric analysis demonstrated that WA covalently modifies NEMO on a cysteine residue within the C-terminal zinc finger (ZF) domain. Point mutations to the ZF can reverse the WA-induced Lys-48-polyubiquitin binding phenotype. Our study demonstrates the feasibility of directly altering the ubiquitin interaction properties of an ubiquitin-binding protein by a chemical compound, thereby shedding light on a novel drug class to potentially alter polyubiquitin-based cellular processes.