L-Tryptophan prevents Escherichia coli biofilm formation and triggers biofilm degradation

The effect of deletion of trp operon and tna operon on the Escherichia coli biofilm formation was investigated in order to elucidate the role of L-tryptophan metabolism in biofilm formation. trp operon deletion mutants ΔtrpC, ΔtrpD and ΔtrpE deficient in L-tryptophan biosynthesis showed higher biofilm formation. In addition, ΔtnaC with increased L-tryptophan degradation activity showed higher biofilm formation. On the contrary, ΔtnaA deletion mutant which lost L-tryptophan degradation activity showed low biofilm formation. From these results, it was suggested that decrease of intracellular L-tryptophan level induced biofilm formation and increase of L-tryptophan repressed biofilm formation. So the effect of the addition of L-tryptophan to the medium on the E. coli biofilm formation was investigated. L-Tryptophan addition at starting culture decreased biofilm formation and furthermore L-tryptophan addition after 16 h culture induced the degradation of preformed biofilm. From the above results, it was suggested that maintenance of high intracellular L-tryptophan concentration prevents E. coli biofilm formation and elevation of intracellular L-tryptophan concentration triggers degradation of matured biofilm.     

Mutants of Lactobacillus plantarum ML11-11 deficient in co-aggregation with yeast exhibited reduced activities of mixed-species biofilm formation

Lactic acid bacteria (LAB) mutants deficient in inter-species co-aggregation with yeast were spontaneously derived from Lactobacillus plantarum ML11-11, a significant mixed-species biofilm former in static co-cultures with budding yeasts. These non-co-aggregative mutants also showed significant decreases in mixed-species biofilm formation. These results suggest the important role of co-aggregation between LAB and yeast in mixed-species biofilm formation. Cell surface proteins obtained by 5 M LiCl extraction from the wild-type cells and non-co-aggregative mutant cells were analyzed by SDS-PAGE. There was an obvious difference in protein profiles. The protein band at 30 kDa was present abundantly in the wild-type cell surface fraction but was significantly decreased in the mutant cells. This band assuredly corresponded to the LAB surface factors that contribute to co-aggregation with yeasts.     

The Nucleotide Sequence and Genome Structure of Mung Bean Yellow Mosaic Geminivirus

Complete nucleotide sequences of the infectious cloned DNA components (DNA 1 and DNA 2) of mung bean yellow mosaic virus (MYMV) were determined. MYMV DNA 1 and DNA 2 consists of 2,723 and 2,675 nucleotides respectively. DNA 1 and DNA 2 have little sequence similarity except for a region of approximately 200 bases which is almost identical in the two molecules. Analysis of open reading frames revealed nine potential coding regions for proteins of mol. wt. > 10,000, six in DNA 1 and three in DNA 2. The nucleotide sequence of MYMV DNA was compared with that of bean golden mosaic virus (BGMV), tomato golden mosaic virus (TGMV) and African cassava mosaic virus (ACMV). The 200-base region common to the two DNAs of each virus had little sequence similarity, except for a highly conserved 33-36 base sequence potentially capable of forming a stable hairpin structure. The potential coding regions in the MYMV DNAs had counterparts in the BGMV, TGMV and ACMV, suggesting an overall similarity in genome organization, except for absence of 1L3 in MYMV DNA 1. The most highly conserved ORFs, MYMV 1R1, BGMV 1R1, TGMV 1R1 and ACMV 1R1, are the putative genes for the coat proteins of MYMV, BGMV, TGMV and ACMV, respectively. MYMV 1L1 has also a high degree of sequence similarity with BGMV 1L1, TGMV 1L1 and ACMV 1L1.     

Basic fibroblast growth factor induces osteoclast formation by reciprocally regulating the production of osteoclast differentiation factor and osteoclastogenesis inhibitory factor in mouse osteoblastic cells.

Basic fibroblast growth factor (bFGF) induced osteoclast formation in co-cultures of mouse spleen cells and osteoblasts. Osteoclastogenesis inhibitory factor (OCIF) and a selective cyclooxygenase-2 (COX-2) inhibitor, NS-398, abolished bFGF-induced osteoclast formation. bFGF did not affect spleen cells, but it did affect osteoblasts, to stimulate osteoclast formation. Northern blot analysis revealed that bFGF up-regulated the expression of osteoclast differentiation factor (ODF) and COX-2 and down-regulated the expression of OCIF in primary osteoblastic cells. NS-398 abolished the increase of ODF mRNA, but it had no effect on the decrease of OCIF mRNA. NS-398 suppressed the binding of (125)I-labeled OCIF to osteoblastic cells treated with bFGF. Enzyme-linked immunosorbent assay showed that bFGF inhibited OCIF production by osteoblastic cells, and the inhibition was not affected by NS-398. We conclude that bFGF induces osteoclast formation by stimulating ODF production through COX-2-mediated prostaglandin synthesis and by suppressing OCIF production through a mechanism independent of prostaglandin synthesis.     

Brefeldin A inhibited activity of the sec7 domain of p200, a mammalian guanine nucleotide-exchange protein for ADP-ribosylation factors.

A brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARF) was purified earlier from bovine brain cytosol. Cloning and expression of the cDNA confirmed that the recombinant protein (p200) is a BFA-sensitive ARF GEP. p200 contains a domain that is 50% identical in amino acid sequence to a region in yeast Sec7, termed the Sec7 domain. Sec7 domains have been identified also in other proteins with ARF GEP activity, some of which are not inhibited by BFA. To identify structural elements that influence GEP activity and its BFA sensitivity, several truncated mutants of p200 were made. Deletion of sequence C-terminal to the Sec7 domain did not affect GEP activity. A protein lacking 594 amino acids at the N terminus, as well as sequence following the Sec7 domain, also had high activity. The mutant lacking 630 N-terminal amino acids was, however, only 1% as active, as was the Sec7 domain itself (mutant lacking 697 N-terminal residues). It appears that the Sec7 domain of p200 contains the catalytic site but additional sequence (perhaps especially that between positions 595 and 630) modifies activity dramatically. Myristoylated recombinant ARFs were better than non-myristoylated as substrates; ARFs 1 and 3 were better than ARF5, and no activity was detected with ARF6. Physical interaction of the Sec7 domain with an ARF1 mutant was demonstrated, but it was much weaker than that of the cytohesin-1 Sec7 domain with the same ARF protein. Effects of BFA on p200 and all mutants with high activity were similar with approximately 50% inhibition at 相似文献   

Suppression of Interleukin-11–mediated bone resorption by cyclooxygenases inhibitors

We previously found that human melanoma (A375M) and human breast cancer (MDA-MB-231) cells formed osteolytic bone metastasis in vivo. These cancer cells produced interleukin-11 (IL-11) by themselves and stimulated its production from osteoblasts. Interleukin-11 could increase the number of osteoclasts and raise the calcium concentration in the medium of neonatal murine calvaria organ culture, indicating bone resorption in vitro. Therefore, IL-11 could play an important role in the promotion of osteolysis at the site of bone metastasis. In the present study, we used the calvaria culture system to try to clarify the mechanisms of IL-11–mediated bone resorption. The murine calvaria expressed both the specificity-determining α subunit and the signal–transducing β subunit (gp130) of the IL-11 receptor. When IL-11 was added to the calvaria culture, the concentrations of prostaglandin E₂ (PGE₂) was elevated. Pretreatment of calvaria with cyclooxygenases inhibitors (e.g., indomethacin, NS-398, and dexamethasone) suppressed the production of PGE₂ and the bone resorption induced by IL-11. Addition of exogenous PGE₂ overcame the inhibitory effect of cyclooxygenases inhibitors and promoted bone resorption. These results indicate that IL-11 promotes bone resorption through a PGE₂ synthesis–dependent mechanism and that cyclooxygenases inhibitors could be interesting drugs to suppress IL-11–mediated osteolytic bone metastasis of cancer cells. J. Cell. Physiol. 175:247–254, 1998. © 1998 Wiley-Liss, Inc.     

Molecular cloning and preliminary expression analysis of banded dogfish (Triakis scyllia) TNF decoy receptor 3 (TNFRSF6B)

Decoy receptor 3 (DcR3), a member of TNF receptor superfamily, is a soluble receptor without death domain and cytoplasmic domain, and secreted by cells and binds with FasL, LIGHT and TL1A. The principal function of DcR3 is the inhibition of apoptosis by the binding cytotoxic ligands. Expression of DcR3 has been reported in a wide array of normal human tissues as well as tumors and tumor cell lines. Recently, DcR3 was reported to modulate a variety of immune responses in mammals. TNFR or DcR3 has been identified in some teleost fishes. However, DcR3 is not reported in cartilaginous fish which is the lowest vertebrate possessing the adaptive immune system. Here we identified DcR3 cDNA in shark (Trsc-DcR3) from an SSH library prepared from peripheral white blood cells stimulated with PMA. Four cysteine-rich domains (CRDs) in common with TNF receptor family members are present in the Trsc-DcR3 sequence. The deduced amino acid sequence of Trsc-DcR3 showed highest identity with the chicken (50.4%), followed by human (46.8%) and rainbow trout (36.5%) DcR3. In a phylogenetic tree of known TNFRSF sequences, the Trsc-DcR3 grouped with the chicken and human DcR3. Trsc-DcR3 mRNA was detected strongly in the gill, moderately in the brain, and weakly in the kidney, thymus and leydig. These data strongly suggest that the gene encoding Trsc-DcR3 in banded dogfish is a homolog of the human gene. mRNA expression of Trsc-DcR3 in the thymus and leydig suggests that DcR3 may act as a modulator in the immune system even at the phylogenetic level of cartilaginous fish.     

Subtilase cytotoxin, produced by Shiga-toxigenic Escherichia coli, transiently inhibits protein synthesis of Vero cells via degradation of BiP and induces cell cycle arrest at G1 by downregulation of cyclin D1

Subtilase cytotoxin (SubAB) is a AB₅ type toxin produced by Shiga-toxigenic Escherichia coli , which exhibits cytotoxicity to Vero cells. SubAB B subunit binds to toxin receptors on the cell surface, whereas the A subunit is a subtilase-like serine protease that specifically cleaves chaperone BiP/Grp78. As noted previously, SubAB caused inhibition of protein synthesis. We now show that the inhibition of protein synthesis was transient and occurred as a result of ER stress induced by cleavage of BiP; it was closely associated with phosphorylation of double-stranded RNA-activated protein kinase-like ER kinase (PERK) and eukaryotic initiation factor-2α (eIF2α). The phosphorylation of PERK and eIF2α was maximal at 30–60 min and then returned to the control level. Protein synthesis after treatment of cells with SubAB was suppressed for 2 h and recovered, followed by induction of stress-inducible C/EBP-homologous protein (CHOP). BiP degradation continued, however, even after protein synthesis recovered. SubAB-treated cells showed cell cycle arrest in G1 phase, which may result from cyclin D1 downregulation caused by both SubAB-induced translational inhibition and continuous prolonged proteasomal degradation.