O-GlcNAc modulation at Akt1 Ser473 correlates with apoptosis of murine pancreatic beta cells

O-GlcNAc transferase (OGT)-mediated modification of protein Ser/Thr residues with O-GlcNAc influences protein activity, similar to the effects of phosphorylation. The anti-apoptotic Akt1 is both activated by phosphorylation and modified with O-GlcNAc. However, the nature and significance of the Akt1 O-GlcNAc modification is unknown. The relationship of O-GlcNAc modification and phosphorylation at Akt1 Ser473 was examined with respect to apoptosis of murine beta-pancreatic cells. Glucosamine treatment induced apoptosis, which correlated with enhanced O-GlcNAc modification of Akt1 and concomitant reduction in Ser473 phosphorylation. Pharmacological inhibition of OGT or O-GlcNAcase revealed an inverse correlation between O-GlcNAc modification and Ser473 phosphorylation of Akt1. MALDI-TOF/TOF mass spectrometry analysis of Akt1 immunoprecipitates from glucosamine-treated cells, but not untreated controls, showed a peptide containing S473/T479 that was presumably modified with O-GlcNAc. Furthermore, in vitro O-GlcNAc-modification analysis of wildtype and mutant Akt1 revealed that S473 was targeted by recombinant OGT. A S473A Akt1 mutant demonstrated reduced basal and glucosamine-induced Akt1 O-GlcNAc modification compared with wildtype Akt1. Furthermore, wildtype Akt1, but not the S473A mutant, appeared to be associated with OGT following glucosamine treatment. Together, these observations suggest that Akt1 Ser473 may undergo both phosphorylation and O-GlcNAc modification, and the balance between these may regulate murine beta-pancreatic cell fate.     

N‐glycan maturation is crucial for cytokinin‐mediated development and cellulose synthesis in Oryza sativa

To explore the physiological significance of N‐glycan maturation in the plant Golgi apparatus, gnt1, a mutant with loss of N‐acetylglucosaminyltransferase I (GnTI) function, was isolated in Oryza sativa. gnt1 exhibited complete inhibition of N‐glycan maturation and accumulated high‐mannose N‐glycans. Phenotypic analyses revealed that gnt1 shows defective post‐seedling development and incomplete cell wall biosynthesis, leading to symptoms such as failure in tiller formation, brittle leaves, reduced cell wall thickness, and decreased cellulose content. The developmental defects of gnt1 ultimately resulted in early lethality without transition to the reproductive stage. However, callus induced from gnt1 seeds could be maintained for periods, although it exhibited a low proliferation rate, small size, and hypersensitivity to salt stress. Shoot regeneration and dark‐induced leaf senescence assays indicated that the loss of GnTI function results in reduced sensitivity to cytokinin in rice. Reduced expression of A‐type O. sativa response regulators that are rapidly induced by cytokinins in gnt1 confirmed that cytokinin signaling is impaired in the mutant. These results strongly support the proposed involvement of N‐glycan maturation in transport as well as in the function of membrane proteins that are synthesized via the endomembrane system.     

TXNIP Deficiency Exacerbates Endotoxic Shock via the Induction of Excessive Nitric Oxide Synthesis

Thioredoxin-interacting protein (TXNIP) has multiple functions, including tumor suppression and involvement in cell proliferation and apoptosis. However, its role in the inflammatory process remains unclear. In this report, we demonstrate that Txnip^−/− mice are significantly more susceptible to lipopolysaccharide (LPS)-induced endotoxic shock. In response to LPS, Txnip^−/− macrophages produced significantly higher levels of nitric oxide (NO) and inducible nitric oxide synthase (iNOS), and an iNOS inhibitor rescued Txnip^−/− mice from endotoxic shock-induced death, demonstrating that NO is a major factor in TXNIP-mediated endotoxic shock. This susceptibility phenotype of Txnip^−/− mice occurred despite reduced IL-1β secretion due to increased S-nitrosylation of NLRP3 compared to wild-type controls. Taken together, these data demonstrate that TXNIP is a novel molecule that links NO synthesis and NLRP3 inflammasome activation during endotoxic shock.     

Protective Efficacy of a Human Endogenous Retrovirus Envelope-Coated,Nonreplicable, Baculovirus-Based Hemagglutin Vaccine against Pandemic Influenza H1N1 2009

Despite the advantages of DNA vaccines, overcoming their lower efficacy relative to that of conventional vaccines remains a challenge. Here, we constructed a human endogenous retrovirus (HERV) envelope-coated, nonreplicable, baculovirus-based HA vaccine against swine influenza A/California/04/2009(H1N1) hemagglutin (HA) (AcHERV-sH1N1-HA) as an alternative to conventional vaccines and evaluated its efficacy in two strains of mice, BALB/c and C57BL/6. A commercially available, killed virus vaccine was used as a positive control. Mice were intramuscularly administered AcHERV-sH1N1-HA or the commercial vaccine and subsequently given two booster injections. Compared with the commercial vaccine, AcHERV-sH1N1-HA induced significantly higher levels of cellular immune responses in both BALB/c and C57BL/6 mice. Unlike cellular immune responses, humoral immune responses depended on the strain of mice. Following immunization with AcHERV-sH1N1-HA, C57BL/6 mice showed HA-specific IgG titers 10- to 100-fold lower than those of BALB/c mice. In line with the different levels of humoral immune responses, the survival of immunized mice after intranasal challenge with sH1N1 virus (A/California/04/2009) depended on the strain. After challenge with 10-times the median lethal dose (MLD₅₀) of sH1N1 virus, 100% of BALB/c mice immunized with the commercial vaccine or AcHERV-sH1N1-HA survived. In contrast, C57BL/6 mice immunized with AcHERV-sH1N1-HA or the commercial vaccine showed 60% and 70% survival respectively, after challenge with sH1N1 virus. In all mice, virus titers and results of histological analyses of lung tissues were consistent with the survival data. Our results indicate the importance of humoral immune response as a major defense system against influenza viral infection. Moreover, the complete survival of BALB/c mice immunized with AcHERV-sH1N1-HA after challenge with sH1N1 virus suggests the potential of baculoviral vector-based vaccines to achieve an efficacy comparable to that of killed virus vaccines.     

Effect of various ions, pH, and osmotic pressure on oxidation of elemental sulfur by Thiobacillus thiooxidans.

The oxidation of elemental sulfur by Thiobacillus thiooxidans was studied at pH 2.3, 4.5, and 7.0 in the presence of different concentrations of various anions (sulfate, phosphate, chloride, nitrate, and fluoride) and cations (potassium, sodium, lithium, rubidium, and cesium). The results agree with the expected response of this acidophilic bacterium to charge neutralization of colloids by ions, pH-dependent membrane permeability of ions, and osmotic pressure.     

Trends in the Rates of Peripartum Hysterectomy and Uterine Artery Embolization

The objective of this study was to determine the trends in national rates of peripartum hysterectomy (PH) and uterine arterial embolization (UAE) in Korea. We used data collected by the Health Insurance Review & Assessment Service of Korea and analyzed data from patients who gave birth during the period from 2005 to 2008. There were 1785,178 deliveries during the study period, including 2636 cases of PH (1.48 per 1000 deliveries). The PH rate in 2005 was 1.57 per 1000 deliveries and in 2008 it was 1.33 per 1000 deliveries. UAE was performed in 161 women (incidence, 0.38 per 1000 deliveries) and 447 women (incidence, 0.98 per 1000 deliveries) in 2005 and 2008, respectively. In Korea, the rate of PH decreased slightly, while the rate of UAE rate increased dramatically during the period from 2005 to 2008. Further studies are needed to evaluate the effects of UAE on the rate of PH performed.     

Colorimetric heparinase assay for alternative anti-metastatic activity

Heparanase has been previously associated with the metastatic potential, inflammation, and angiogenesis of tumor cells. Heparanase activity has been detected by means of UV absorption, radiolabeled substrates, electrophoretic migration, and heparan sulfate affinity assays. However, those methods have proven to be somewhat problematic with regards to application to actual biological samples, the accessibility of the immobilized substrates, experimental sensitivity, and the separation of degraded products. Rather than focusing on heparanase activity, then, we have developed a rapid, alternative colorimetric heparinase assay, on the basis of the recent finding that sulfated disaccharides generated from heparin by bacterial heparinase exhibit biological properties comparable to those from heparan sulfate by mammalian heparanase. In this study, the concentrations of porcine heparin and bacterial heparinase I were determined using a Sigma Diagnostics Kit. Morus alba was selected as a candidate through this assay system, and an inhibitor, resveratrol, was purified from its methanol extract. Its anti-metastatic effects on the pulmonary metastasis of murine B16 melanoma cells were also evaluated. Our findings suggest that this assay may prove useful as a diagnostic tool for heparinase inhibition, as an alternative anti-metastatic target.     

Light activates the degradation of PIL5 protein to promote seed germination through gibberellin in Arabidopsis

Angiosperm seeds integrate various environmental signals, such as water availability and light conditions, to make a proper decision to germinate. Once the optimal conditions are sensed, gibberellin (GA) is synthesized, triggering germination. Among environmental signals, light conditions are perceived by phytochromes. However, it is not well understood how phytochromes regulate GA biosynthesis. Here we investigated whether phytochromes regulate GA biosynthesis through PIL5, a phytochrome-interacting bHLH protein, in Arabidopsis. We found that pil5 seed germination was inhibited by paclobutrazol, the ga1 mutation was epistatic to the pil5 mutation, and the inhibitory effect of PIL5 overexpression on seed germination could be rescued by exogenous GA, collectively indicating that PIL5 regulates seed germination negatively through GA. Expression analysis revealed that PIL5 repressed the expression of GA biosynthetic genes (GA3ox1 and GA3ox2), and activated the expression of a GA catabolic gene (GA2ox) in both PHYA- and PHYB-dependent germination assays. Consistent with these gene-expression patterns, the amount of bioactive GA was higher in the pil5 mutant and lower in the PIL5 overexpression line. Lastly, we showed that red and far-red light signals trigger PIL5 protein degradation through the 26S proteasome, thus releasing the inhibition of bioactive GA biosynthesis by PIL5. Taken together, our data indicate that phytochromes promote seed germination by degrading PIL5, which leads to increased GA biosynthesis and decreased GA degradation.     

Partial characterization and cloning of leuconocin J, a bacteriocin produced by Leuconostoc sp. J2 isolated from the Korean fermented vegetable Kimchi

Leuconostoc sp. J2, isolated from naturally fermented Kimchi, produced a bacteriocin which was named leuconocin J. This bacteriocin exhibited an inhibitory activity against several lactic acid bacteria and some food-borne pathogens. The antimicrobial substance was secreted into the medium during the late log phase. It appears to be proteinaceous since its activity was completely inactivated by a range of proteolytic enzymes, and it was also relatively heat-stable. The bacteriocin was partially purified by ammonium sulphate precipitation, following dialysis. The apparent molecular mass of partially purified bacteriocin, as indicated by activity detection after Tricine-SDS-PAGE, was 2.5-3.5 kDa. Leuconostoc sp. J2 plasmid DNA digested by EcoRI was cloned into pUC118 and transformed into Escherichia coli DH5 alpha. Phenotypic expression of the bacteriocin production was detected in transformants harboring pULBJ5.5. Finally, Southern blotting with the 2.3 kb insert as a probe against plasmid digests of Leuconostoc sp. J2 revealed that the cloned foreign DNA originated from Leuconostoc sp. J2.