Over-expression of transglutaminase in the Drosophila eye imaginal disc induces a rough eye phenotype

Transglutaminases (TGs) catalyze the cross-linking of proteins and are involved in various biological processes in mammals. In invertebrates, except for the involvement in the hemolymph clotting, the functions of TG have not been revealed. Drosophila has a single TG gene (CG7356), from which two kinds of mRNAs (dTG-RA and dTG-RB) are formed. RT-PCR analyses indicated that both dTGs-RA and -RB are synthesized in all the developmental stages tested. To reveal the roles of dTG during the development, we examined a phenotype induced through the ectopic expression of dTG by using a GAL4-UAS targeted expression system. Over-expression of dTG-A in the eye imaginal disc of larva induced a rough eye phenotype in adult compound eyes. Co-expression of P35, an inhibitor of apoptosis, suppressed the rough eye phenotype, suggesting that the rough eye phenotype induced by the over-expression of dTG-A in the eye imaginal disc is due to the occurrence of apoptosis. The rough eye phenotype induced by the over-expression of dTG-A was suppressed by the crossing with mutant fly lines lacking Drosophila JNK gene basket (bsk) or Drosophila JNKK gene hemipterous. FLP-out experiments using an enhancer trap line showed that the over-expression of dTG-A in the eye imaginal disc increased the puckered enhancer activity, a reporter of Bsk activity. These results suggested that the rough eye phenotype induced by the over-expression of dTG-A is related to an enhancement of JNK signaling pathway.     

High-Temperature-Induced Transposition of Insertion Elements in Burkholderia multivorans ATCC 17616

An efficient and quantitative method to analyze the transposition of various insertion sequence (IS) elements in Burkholderia multivorans ATCC 17616 was devised. pGEN500, a plasmid carrying a Bacillus subtilis-derived sacB gene, was introduced into ATCC 17616 cells, and 25% of their sucrose-resistant derivatives were found to carry various IS elements on pGEN500. A PCR-based experimental protocol, in which a mixture of several specific primer pairs was used, revealed that pGEN500 captured, in addition to five previously reported IS elements (IS401, IS402, IS406, IS407, and IS408), three novel IS elements, ISBmu1, ISBmu2, and ISBmu3. The global transposition frequency of these IS elements was enhanced more than sevenfold under a high-temperature condition (42°C) but not under oxidative stress or starvation conditions. To our knowledge, this is the first report demonstrating the elevated transposition activities of several IS elements at a high temperature. The efficient experimental protocol developed in this study will be useful in quantitatively and simultaneously investigating various IS elements, as well as in capturing novel functional mobile elements from a wide variety of bacteria.     

Mechanism of growth inhibitory effect of Blumea balsamifera extract in hepatocellular carcinoma

Blumea balsamifera is known to improve physiological disorders such as rheumatism and hypertension, but its anticancer activity has not been well elucidated. In this study, we found that Blumea balsamifera MeOH extract (BME) induced growth-inhibitory activity in rat and human hepatocellular carcinoma cells without cytotoxicity in rat hepatocytes which were used as a normal cell model. BME induced cell cycle arrest at the G1 phase via decreases in the expression of cyclin-E and phosphorylation of retinoblastoma protein. Furthermore, BME reduced the level of a proliferation-inducing ligand, that stimulates tumor cell growth. These findings suggest that BME has possible therapeutic potential in hepatoma cancer patients and that depletion of cellular APRIL is an important mechanism in the growth-inhibitory effect of BME.     

Glial Fibrillary Acidic Protein is Greatly Modified by Oxidative Stress in Aceruloplasminemia Brain

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism caused by mutations in the ceruloplasmin (Cp) gene. The neuropathological hallmark of this disease is intracellular iron overload, which is thought to lead to neuronal cell death through increased oxidative stress. We evaluated and characterized protein oxidation in the brain of a patient with this disease. The protein carbonyl content in the cerebral cortex of the patient was elevated compared to controls. Furthermore, peptide mass fingerprinting and partial amino acid sequencing identified glial fibrillary acidic protein (GFAP) as the major carbonylated protein in the cerebral cortex of the patient. In conjunction with the facts that Cp mainly localizes to astrocytes in the central nervous system and that astrocytes are loaded with much more iron than neurons in the cerebral cortex, our findings indicate that Cp deficiency may primarily damage astrocytes. We speculate that the dysfunction of astrocytes may be causatively related to neuronal cell loss in aceruloplasminemia.     

Membrane-associated Activation of Cholesterol ��-Glucosyltransferase, an Enzyme Responsible for Biosynthesis of Cholesteryl-��-d-Glucopyranoside in Helicobacter pylori Critical for Its Survival

Helicobacter pylori (H. pylori) is the causative pathogen underlying gastric diseases such as chronic gastritis and gastric cancer. Previously, the authors revealed that α1,4-linked N-acetylglucosamine-capped O-glycan (αGlcNAc) found in gland mucin suppresses H. pylori growth and motility by inhibiting catalytic activity of cholesterol α-glucosyltransferase (CHLαGcT), the enzyme responsible for biosynthesis of the major cell wall component cholesteryl-α-d-glucopyranoside (CGL). Here, the authors developed a polyclonal antibody specific for CHLαGcT and then undertook quantitative ultrastructural analysis of the enzyme’s localization in H. pylori. They show that 66.3% of CHLαGcT is detected in the cytoplasm beneath the H. pylori inner membrane, whereas 24.7% is present on the inner membrane. In addition, 2.6%, 5.0%, and 1.4% of the protein were detected in the periplasm, on the outer membrane, and outside microbes, respectively. By using an in vitro CHLαGcT assay with fractionated H. pylori proteins, which were used as an enzyme source for CHLαGcT, the authors demonstrated that the membrane fraction formed CGL, whereas other fractions did not. These data combined together indicate that CHLαGcT is originally synthesized in the cytoplasm of H. pylori as an inactive form and then activated when it is associated with the cell membrane. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.     

Effects of modern eating patterns on the cardiac autonomic nervous system in young Japanese males

The aim of this study was to determine the effects of the single intake of a high-energy and high-fat meal, of that of a moderate-energy and high-carbohydrate meal, and of fasting, which are major global eating patterns involving the combination of various levels of energy and nutrients, on heart rate variability in healthy young males. Participants were assigned to three groups: the high-energy and high-fat meal group, the moderate-energy and high-carbohydrate meal group, and the fasting group (no meal) in a randomized crossover design. The R-R intervals were continuously recorded before and after meals. Physiological and psychological data were obtained before and 30, 60, 90, and 120 min after meal intake. The main results were: (1) decreased sympathetic modulation of the heart and increased parasympathetic modulation of the heart in the fasting group, indicated by an unchanged heart rate, a decreased lowfrequency/high-frequency ratio, and increased high-frequency power of heart rate variability in the fasting group; (2) cardiac sympathetic activation or parasympathetic withdrawal after the intake of either a high-energy and high-fat meal or a moderate-energy and high-carbohydrate meal, indicated by increases in the heart rate and the low-frequency/high-frequency ratio of heart rate variability, and a decrease in the high-frequency power of heart rate variability in both the high-energy and high-fat meal group and the moderate-energy and high-carbohydrate meal group; and (3) the high-energy and high-fat meal group and the moderate-energy and high-carbohydrate meal group showed similar movement in physiological and psychological measurements after the meal intake. In conclusion, the intake of the high-energy and high-fat meal and the moderate-energy and high-carbohydrate meal similarly activated sympathetic modulation of the heart, whereas fasting suppressed sympathetic modulation of the heart compared with the other two eating groups in healthy young males.     

IGF-1 promotes β-amyloid production by a secretase-independent mechanism

β-Amyloid peptide (Aβ) is generated via the sequential proteolysis of β-amyloid precursor protein (APP) by β- and γ-secretases, and plays a crucial role in the pathogenesis of Alzheimer’s disease (AD). Here, we sought to clarify the role of insulin-like growth factor-1 (IGF-1), implicated in the AD pathomechanism, in the generation of Aβ. Treatment of neuroblastoma SH-SY5Y cells expressing AD-associated Swedish mutant APP with IGF-1 did not alter cellular levels of APP, but significantly increased those of β-C-terminal fragment (β-CTF) and secreted Aβ. IGF-1 also enhanced APP phosphorylation at Thr668. Treatment of β-CTF-expressing cells with IGF-1 increased the levels of β-CTF and secreted Aβ. The IGF-1-induced augmentation of β-CTF was observed in the presence of γ-secretase inhibitors, but not in cells expressing β-CTF with a Thr668 to alanine substitution. These results suggest that IGF-1 promotes Aβ production through a secretase-independent mechanism involving APP phosphorylation.     

Epitope-tagged L* protein of Theiler's murine encephalomyelitis virus is expressed in the central nervous system in the acute phase of infection

TO subgroup strains of Theiler's murine encephalomyelitis virus (TMEV) synthesize L* protein from an alternative initiation codon. We first demonstrated L* expression in the central nervous system (CNS) of TMEV-infected mice during the acute phase of infection by immunoprecipitation and immunoblotting with anti-L* antibody. In addition, we generated mutant viruses which synthesize FLAG or 3xFLAG epitope-tagged L* protein. With a mutant virus expressing 3xFLAG epitope-tagged L*, designated DA/3xFLAGL*, we investigated L* in the CNS in the acute phase of infection. DA/3xFLAGL* did not change the virus tropism in comparison with wild-type virus, and L* was clearly identified in the CNS in both susceptible and resistant strains of mice. Double immunolabeling studies showed that L* is colocalized with TMEV polyprotein and exclusively expressed in neurons.