A novel multiplex PCR method for Clostridium botulinum neurotoxin type A gene cluster typing

A rapid, simple and sensitive multiplex PCR method for boNT/A gene cluster typing was developed by combining the results of BoNT/A subtype (boNT/A1 or /A2) gene detection with ha33 and/or p47 gene detection. Ten isolates associated with infant botulism in Japan were examined and divided into boNT/A gene cluster types 2 and 3 by origin (honey feeding or not) and period (1986–1987 or 1999–2007). It is suggested that this multiplex PCR method will be be useful for epidemiological studies of botulism.     

cAMP-dependent Protein Kinase and c-Jun N-terminal Kinase Mediate Stathmin Phosphorylation for the Maintenance of Interphase Microtubules during Osmotic Stress

Dynamic microtubule changes after a cell stress challenge are required for cell survival and adaptation. Stathmin (STMN), a cytoplasmic microtubule-destabilizing phosphoprotein, regulates interphase microtubules during cell stress, but the signaling mechanisms involved are poorly defined. In this study ectopic expression of single alanine-substituted phospho-resistant mutants demonstrated that STMN Ser-38 and Ser-63 phosphorylation were specifically required to maintain interphase microtubules during hyperosmotic stress. STMN was phosphorylated on Ser-38 and Ser-63 in response to hyperosmolarity, heat shock, and arsenite treatment but rapidly dephosphorylated after oxidative stress treatment. Two-dimensional PAGE and Phos-tag gel analysis of stress-stimulated STMN phospho-isoforms revealed rapid STMN Ser-38 phosphorylation followed by subsequent Ser-25 and Ser-63 phosphorylation. Previously, we delineated stress-stimulated JNK targeting of STMN. Here, we identified cAMP-dependent protein kinase (PKA) signaling as responsible for stress-induced STMN Ser-63 phosphorylation. Increased cAMP levels induced by cholera toxin triggered potent STMN Ser-63 phosphorylation. Osmotic stress stimulated an increase in PKA activity and elevated STMN Ser-63 and CREB (cAMP-response element-binding protein) Ser-133 phosphorylation that was substantially attenuated by pretreatment with H-89, a PKA inhibitor. Interestingly, PKA activity and subsequent phosphorylation of STMN were augmented in the absence of JNK activation, indicating JNK and PKA pathway cross-talk during stress regulation of STMN. Taken together our study indicates that JNK- and PKA-mediated STMN Ser-38 and Ser-63 phosphorylation are required to preserve interphase microtubules in response to hyperosmotic stress.     

Inhibition of Cell Proliferation and Migration by miR-509-3p That Targets CDK2, Rac1, and PIK3C2A

CDK2 is a key regulator of cell cycle progression. In this study, we screened for miRNAs targeting CDK2 using a luciferase-3′-untranslated region reporter assay. Among 11 hit miRNAs, miR-509-3p reduced CDK2 protein levels and significantly inhibited cancer cell growth. Microarray, Western blotting, and luciferase reporter analyses revealed additional targets of miR-509-3p, including Rac1 and PIK3C2A. Overexpression of miR-509-3p induced G1 cell-cycle arrest and inhibited colony formation and migration. RNAi experiments indicated that the growth-inhibitory effects of miR-509-3p may occur through down-regulation of CDK2, Rac1, and PIK3C2A. Targeting of multiple growth regulatory genes by miR-509-3p may contribute to effective anti-cancer therapy.     

The Human Enzyme That Converts Dietary Provitamin A Carotenoids to Vitamin A Is a Dioxygenase

β-Carotene 15–15′-oxygenase (BCO1) catalyzes the oxidative cleavage of dietary provitamin A carotenoids to retinal (vitamin A aldehyde). Aldehydes readily exchange their carbonyl oxygen with water, making oxygen labeling experiments challenging. BCO1 has been thought to be a monooxygenase, incorporating oxygen from O₂ and H₂O into its cleavage products. This was based on a study that used conditions that favored oxygen exchange with water. We incubated purified recombinant human BCO1 and β-carotene in either ¹⁶O₂-H₂¹⁸O or ¹⁸O₂-H₂¹⁶O medium for 15 min at 37 °C, and the relative amounts of ¹⁸O-retinal and ¹⁶O-retinal were measured by liquid chromatography-tandem mass spectrometry. At least 79% of the retinal produced by the reaction has the same oxygen isotope as the O₂ gas used. Together with the data from ¹⁸O-retinal-H₂¹⁶O and ¹⁶O-retinal-H₂¹⁸O incubations to account for nonenzymatic oxygen exchange, our results show that BCO1 incorporates only oxygen from O₂ into retinal. Thus, BCO1 is a dioxygenase.     

Isolation of Cell Surface Membranes from Cultured C6 Glioblastoma Cells

Abstract: Plasma membranes were isolated from C6 glioblastoma cells by two methods. In the first method cells were treated with concanavalin A and lysed in hypotonic medium. After partial separation of plasma membranes from other cell material, the lectin was displaced with a-methyl-D-mannoside. In the second method untreated cells or cells iodinated in a lactoperoxidase-catalyzed reaction were homogenized in isotonic medium. Membrane fractions obtaincd by either homogenization procedure were further purified by rate zonal and equilibrium centrifugations into linear density gradients. Disruption of the glioblastoma cell membrane gives rise to heterogeneous assemblies of mem- brane fragments. Two populations of plasma membranes were isolated from untreated and from iodinated cells: a "lighter")membrane fraction characterized by relatively lower sedimentation velocity and buoyant density, and a "heavier" membrane fraction of relatively faster sedimentation velocity and higher buoyant density. Both fractions showed electrophoretic patterns similar to those of ¹²⁵I-labeled cell surface proteins. Their specific (Na⁺+ K⁺)-ATPase activity was seven- to eightfold the homogenate activity (recovery, 13.1%). Both fractions were, however, still contaminated by smooth endo- plasmic reticulum, as judged from the activity 0: NADPH-dependent cytochrome c reductase (recovery, 2.4%). It is suggested that plasma membrane fragments present in the two fractions might differ in the organization of their structures, e.g., membrane vesicle intactness and membrane orientation.     

Lectin involvement in root-hair tip adhesion as related to the Rhizobium-clover symbiosis

Contact of adjacent root hairs of seedlings of white clover ( Trifolium repens L. cv. Ladino and Louisiana Nolin) led to cell-cell adhesion of root hair tips. The involvement of the root lectin, trifoliin A, in this phenomen was examined in slide cultures of axenically grown seedlings. Trifoliin A was detected by indirect immunofluorescence on root hair tips, which had adhered to one another. Seedlings grown under conditions which specifically reduce the levels of this lectin on the root surface (e.g., in the presence of 15 m M NO₃– or 5 m M 2-deoxy- d -glucose) had significantly fewer adhesions of root hair tips. In addition, flushing the slide cultures with 20 m M 2-deoxy- d -glucose resulted in an immediate 4-fold reduction in frequency of tip adhesions. These results are consistent with the lectin cross-bridging model, which predicts that cell-cell adhesions would occur when trifoliin A on root hair tips contacts complementary glycosylated receptors on neighboring root hairs.