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We constructed a new Thermus thermophilus cloning vector which enables the colour selection of cloned DNA inserts in the T. thermophilus HB27 host strain (β-gal) on growth plates containing 3,4-cyclohexenoesculetin β- d-galactopyranoside (S-gal) in the medium. This vector harbors a modified β-galactosidase gene (TTP0042 of T. thermophilus HB27) with 12 unique restriction enzyme sites ( Acc65I, AvrII, BlpI, BssHII, EcoRI, EcoRV, HindIII, NruI, SalI, SpeI, SphI and XbaI) as multiple cloning sites under the control of the T. thermophilus slpA promoter. This host-vector system facilitates cloning procedures in T. thermophilus HB27. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-α (TNFα)-induced reactions are effective to maintain homeostasis; however, excessive responses play progressive roles in the pathogenesis of various chronic inflammatory diseases. We demonstrate that TNFα triggered the release of its receptor TNFR1 as a content of extracellular vesicles (EVs) from the human bronchial epithelial cell, BEAS-2b. The TNFR1 cytoplasmic domain binding partner, TNFR-associated death domain (TRADD), was released by TNFα treatment along with TNFR1. TNFα-triggered release of EVs was decreased in the presence of amitriptyline, an inhibitor of acid sphingomyelinase (A-SMase), or of GW4869, an inhibitor of neutral sphingomyelinase (N-SMase), indicating that EVs containing TNFR1 and TRADD are released through A-SMase and N-SMase dependent manners. From sucrose density gradient analysis, each sphingomyelinase is involved in the generation of distinct populations of EVs. Inhibition of A-SMase or N-SMase resulted in significantly increased responses to TNFα in parental cells. Given that TRADD serves as a platform for the assembly of subsequent signaling molecules, the TNFα triggered release of TNFR1 and TRADD might be an effective strategy for down regulation of the TNFα responses of parental cells. [ABSTRACT FROM AUTHOR]

p230/golgin-245 is a trans -Golgi coiled-coil protein that is known to participate in regulatory transport from the trans -Golgi network (TGN) to the cell surface. We investigated the role of p230 and its interacting protein, microtubule actin crosslinking protein 1 (MACF1), in amino acid starvation-induced membrane transport. p230 or MACF1 knock-down (KD) cells failed to increase the autophagic flow rate and the number of microtubule-associated protein 1 light chain 3 (LC3)-positive puncta under starvation conditions. Loss of p230 or MACF1 impaired mAtg9 recruitment to peripheral phagophores from the TGN, which was observed in the early step of autophagosome formation. Overexpression of the p230-binding domain of MACF1 resulted in the inhibition of mAtg9 trafficking in starvation conditions as in p230-KD or MACF1-KD cells. These results indicate that p230 and MACF1 cooperatively play an important role in the formation of phagophore through starvation-induced transport of mAtg9-containing membranes from the TGN. In addition, p230 itself was detected in autophagosomes/autolysosome with p62 or LC3 during autophagosome biogenesis. Thus, p230 is an important molecule in phagophore formation, although it remains unclear whether p230 has any role in late steps of autophagy. [ABSTRACT FROM AUTHOR]

We developed a reporter gene system that enables precise analysis of promoter activity in Thermus thermophilus HB27. The reporter vector employs a promoterless β-galactosidase gene of Thermus spp. strain T2. However, T. thermophilus HB27 strain has three genes (TTP0042, TTP0220 and TTP0222) whose products have β-galactosidase activity, which would interfere with correct measurements of promoter activities. Thus, to eliminate this background activity, we disrupted all three of these genes to generate a host strain for measuring promoter expression as β-galactosidase activity. In addition, T. thermophilus strains also produce carotenoids called thermoxanthins that are yellow pigments. To avoid the influence of these carotenoids on the β-galactosidase assay, we also disrupted the phytoene synthase gene ( crtB). The reporter gene system developed here is a powerful tool for studying transcriptional activity and the mechanisms that regulate gene expression in T. thermophilus HB27. We also showed that the crtB gene cassette could be used in repeated gene-disruption experiments to screen transformants by colony colour, thus eliminating the need for antibiotic resistance markers. [ABSTRACT FROM AUTHOR]

GM130 is a cytoplasmic peripheral membrane protein localized on the cis side of the Golgi apparatus. GM130 is proposed to function as a membrane skeleton, maintaining the structure of the Golgi apparatus, and as a vesicle tether that facilitates vesicle fusion to the Golgi membrane. More than 60% of the GM130 molecule is believed to exist as coiled-coil structures with a probability above 90%, based on its primary amino acid sequence. The predicted coiled-coil region was similar to that of yeast Uso1p and its mammalian homolog, p115, both of which form coiled-coil homodimers. Therefore, GM130 has long been thought to form a homodimer with a rod-like shape. However, our biochemical and electron microscopical analyses revealed that GM130 is a parallel homotetramer with a flexible rod-like structure with I- and Y-shaped conformations. The structure of the N-terminal region may interchange between an open conformation (branched or Y-shaped) and a closed conformation (non-branched or I-shaped), possibly with the help of interacting molecules. This conformational change may alter the oligomeric state of the GM130 molecules and the function of GM130 in the vesicle tethering and the maintenance of the Golgi structure. [ABSTRACT FROM AUTHOR]