Probabilistic transition from unstable predator-prey interaction to stable coexistence of Dictyostelium discoideum and Escherichia coli.

Predator-prey interactions have been found at all levels within ecosystems. Despite their ecological ubiquity and importance, the process of transition to a stable coexistent state has been poorly verified experimentally. To investigate the stabilization process of predator-prey interactions, we previously constructed a reproducible experimental predator-prey system between Dictyostelium discoideum and Escherichia coli, and showed that the phenotypically changed E.

Membrane topology and functional importance of the periplasmic region of ABC transporter LolCDE.

The LolCDE complex is an ATP-binding cassette transporter that mediates the release of newly synthesized lipoproteins from the cytoplasmic membrane of gram-negative bacteria, which results in the initiation of outer-membrane sorting of lipoproteins through the Lol pathway. LolCDE is composed of one copy each of membrane subunits LolC and LolE, and two copies of nucleotide-binding subunit LolD. In this study, we examined the membrane topology of LolC and LolE by PhoA fusion analysis.

Mitomycin-C treatment followed by culture produces long-term survival of islet xenografts in a rat-to mouse model.

One of the goals of islet transplantation is to transplant viable islets without host immunosuppression. The present study was designed to determine whether pretreatment of islets with mitomycin-C (MMC) followed by culture enhances islet survival in a rat-to-mouse xenogeneic combination. WS(RT1k) rat islets pretreated with various concentrations of MMC (0, 3.

Phenotypic plasticity of Escherichia coli at initial stage of symbiosis with Dictyostelium discoideum.

We observed the change in the physiological state of Escherichia coli cells at the initial stage for establishing a new symbiotic relationship with Dictyostelium discoideum cells. For the physiological state, we monitored green fluorescence intensity due to a green fluorescent protein (GFP) gene integrated into the chromosome by flow cytometry (FCM). On co-cultivation of the two species, a new population of E.

A new screening method to identify inhibitors of the Lol (localization of lipoproteins) system, a novel antibacterial target.

As the Lol system, which is involved in localization of lipoproteins, is essential for Escherichia coli growth and widely conserved among gram-negative bacteria, it is considered to be a promising target for the development of anti-gram-negative bacterial agents. However, no high-throughput screening method has so far been developed to screen for Lol system inhibitors. By combining three assay systems (anucleate cell blue assay, Lpp assay, and LolA-dependent release inhibition assay) and a drug susceptibility test, we have successfully developed a new screening method for identification of compounds that inhibit the Lol system.

Genetic analysis of the mode of interplay between an ATPase subunit and membrane subunits of the lipoprotein-releasing ATP-binding cassette transporter LolCDE.

The LolCDE complex, an ATP-binding cassette (ABC) transporter, releases lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane of Escherichia coli. The LolCDE complex is composed of two copies of an ATPase subunit, LolD, and one copy each of integral membrane subunits LolC and LolE. LolD hydrolyzes ATP on the cytoplasmic side of the inner membrane, while LolC and/or LolE recognize and release lipoproteins anchored to the periplasmic leaflet of the inner membrane.

Roles of the hydrophobic cavity and lid of LolA in the lipoprotein transfer reaction in Escherichia coli.

LolA, a periplasmic chaperone, binds to outer membrane-specific lipoproteins released from the inner membrane through the action of an ATP-binding cassette transporter, LolCDE and then transfers them to the outer membrane receptor LolB, thereby mediating the inner to outer membrane transport of lipoproteins. The crystal structure of free LolA revealed that it has an internal hydrophobic cavity, which is surrounded by hydrophobic residues and closed by a lid comprising alpha-helices. The hydrophobic cavity most likely represents the binding site for the lipid moiety of a lipoprotein.

Understanding the response of dendritic cells to activation by streptococcal preparation OK-432.

Background: The streptococcal preparation OK-432 induces maturation of T cells and dendritic cells (DCs). However, the mechanisms by which OK-432 induces DC maturation are not well understood.

Materials And Methods: The effects of OK-432 and TNF-alpha on peripheral blood mononuclear cells (PBMCs) were compared.

Disruption of rpmJ encoding ribosomal protein L36 decreases the expression of secY upstream of the spc operon and inhibits protein translocation in Escherichia coli.

The spc operon of Escherichia coli encodes 11 ribosomal proteins and SecY. The secY gene and downstream rpmJ encoding a ribosomal protein, L36, are located distal to the promoter of the spc operon. It has been suggested that the stability of SecY mRNA depends on rpmJ unless a rho-independent terminator is inserted immediately downstream of secY.

Mechanisms underlying energy-independent transfer of lipoproteins from LolA to LolB, which have similar unclosed {beta}-barrel structures.

The Lol system, comprising five Lol proteins, transfers lipoproteins from the inner to the outer membrane of Escherichia coli. Periplasmic LolA accepts lipoproteins from LolCDE in the inner membrane and immediately transfers them to LolB, a receptor anchored to the outer membrane. The unclosed beta-barrel structures of LolA and LolB are very similar to each other and form hydrophobic cavities for lipoproteins.

Sorting of lipoproteins to the outer membrane in E. coli.

Escherichia coli lipoproteins are anchored to the periplasmic surface of the inner or outer membrane depending on the sorting signal. An ATP-binding cassette (ABC) transporter, LolCDE, releases outer membrane-specific lipoproteins from the inner membrane, causing the formation of a complex between the released lipoproteins and the periplasmic molecular chaperone LolA. When this complex interacts with outer membrane receptor LolB, the lipoproteins are transferred from LolA to LolB and then localized to the outer membrane.

[Experimental study for a combination chemo-immunotherapy using dendritic cells].

This study was designed to seek for the optimal anticancer agents for a combination of chemotherapy and specific immunotherapy using dendritic cells (DC) in gastric cancer. We investigated the immuno-suppressive activity of anticancer agents on human peripheral blood mononuclear cells (PBMC), apoptosis inducing activity on gastric cancer cells and expression of Toll-like receptor (TLR)-4 mRNA on immatureDCs (iDCs) by paclitaxel (TXL) and docetaxel (TXT). We further compared the cytotoxicity of cytotixic T lymphocytes (CTLs) induced by DCs pulsed with tumor cell lysate and apoptotic cells induced by TXT.

Targeted mutagenesis of five conserved tryptophan residues of LolB involved in membrane localization of Escherichia coli lipoproteins.

LolB, catalyzing the last step of lipoprotein transfer from the inner to the outer membrane of Escherichia coli, is itself a lipoprotein anchored to the outer membrane. Five Trp residues of LolB are conserved among LolB homologs in Gram-negative bacteria. These Trp residues were mutagenized to obtain defective LolB mutants.

Sorting of lipoproteins to the outer membrane in E. coli.

Escherichia coli lipoproteins are anchored to the periplasmic surface of the inner or outer membrane depending on the sorting signal. An ATP-binding cassette (ABC) transporter, LolCDE, releases outer membrane-specific lipoproteins from the inner membrane, causing the formation of a complex between the released lipoproteins and the periplasmic molecular chaperone LolA. When this complex interacts with outer membrane receptor LolB, the lipoproteins are transferred from LolA to LolB and then localized to the outer membrane.

Effects of lipoprotein overproduction on the induction of DegP (HtrA) involved in quality control in the Escherichia coli periplasm.

Recent biochemical examination has revealed the presence of at least 90 different lipoproteins in Escherichia coli. Among previously identified lipoproteins, only an outer membrane lipoprotein, NlpE, is known to induce expression of the degP gene upon its overproduction. The degP gene encodes a periplasmic protease, which is thought to be involved in the digestion of unfolded proteins, and is essential for growth at high temperatures.

Lipoprotein trafficking in Escherichia coli.

Bacterial lipoproteins comprise a subset of membrane proteins that are covalently modified with lipids at the amino-terminal Cys. Lipoproteins are involved in a wide variety of functions in bacterial envelopes. Escherichia coli has more than 90 species of lipoproteins, most of which are located on the periplasmic surface of the outer membrane, while others are located on that of the inner membrane.

Survival of mitomycin C-treated pancreatic islet xenografts is mediated by increased expression of transforming growth factor-beta.

Background: Mitomycin C (MMC) can trigger various intracellular signals. The authors previously showed that pretreatment of highly immunogenic crude pancreatic islets with MMC improved their survival in a rat-to-mouse transplantation model. The aim of this study was to investigate the role of transforming growth factor (TGF)-beta in mediating MMC-induced survival of islet xenografts.

Global change in Escherichia coli gene expression in initial stage of symbiosis with Dictyostelium cells.

Genome-wide gene expression profiling was performed to investigate the early formation of symbiosis using an artificial symbiosis of Escherichia coli and Dictyostelium discoideum. We have previously reported that when these two species were allowed to grow on minimal agar plates, they achieved a stable state of coexistence, in which the emerging E. coli colonies housing Dictyostelium cells were of a mucoidal nature that was not observed originally.

Universality and flexibility in gene expression from bacteria to human.

Highly parallel experimental biology is offering opportunities to not just accomplish work more easily, but to explore for underlying governing principles. Recent analysis of the large-scale organization of gene expression has revealed its complex and dynamic nature. However, the underlying dynamics that generate complex gene expression and cellular organization are not yet understood.

Mechanism underlying the inner membrane retention of Escherichia coli lipoproteins caused by Lol avoidance signals.

Escherichia coli lipoproteins are localized to either the inner or outer membrane depending on the residue at position 2. The inner membrane retention signal, Asp at position 2 in combination with certain residues at position 3, functions as a Lol avoidance signal, i.e.

A practical phasing procedure using the MAD method without the aid of XAFS measurements: successful solution in the structure determination of the outer-membrane lipoprotein carrier LolA.

A practical procedure for MAD phasing was successfully performed in the structure determination of the LolA protein, even though the XAFS data could not be measured owing to the overlap of the fluorescence spectra of the atoms that contribute significant signals for anomalous dispersion. The LolA protein, a periplasmic chaperone functioning as an outer-membrane lipoprotein carrier of the Lol system which mediates translocation of the water-insoluble outer-membrane lipoprotein across the periplasm in Gram-negative bacteria, was crystallized in two forms: orthorhombic (I222) and trigonal (P3(1)21 or P3(2)21). A multi-wavelength data set was collected from a platinum derivative of the orthorhombic crystals grown from a buffer solution containing zinc acetate and cacodylate (an arsenic compound), but XAFS measurements could not be performed because the energies of the fluorescence spectra of Zn atoms, As atoms and Pt atoms are in very close proximity.

Permanent acceptance of mitomycin C-treated islet allograft.

Background: Mitomycin C (MMC) treatment produces genotoxic stress and exerts various biologic effects on cell function. This study determines the feasibility of MMC pretreatment of islet grafts as a sole immunomodulatory regimen to protect murine crude-digested islet allografts.

Methods: Collagenase-digested BALB/c (H-2d) islets were incubated for 30 min with MMC at different doses (0, 3.

Crystal structures of bacterial lipoprotein localization factors, LolA and LolB.

Lipoproteins having a lipid-modified cysteine at the N-terminus are localized on either the inner or the outer membrane of Escherichia coli depending on the residue at position 2. Five Lol proteins involved in the sorting and membrane localization of lipoprotein are highly conserved in Gram-negative bacteria. We determined the crystal structures of a periplasmic chaperone, LolA, and an outer membrane lipoprotein receptor, LolB.