Membrane topology of ABC-type macrolide antibiotic exporter MacB in Escherichia coli

MacB is an ABC-type membrane protein that exports only macrolide compounds containing 14- and 15-membered lactones, cooperating with a membrane fusion protein, MacA, and a multifunctional outer membrane channel, TolC. We determined the membrane topology of MacB by means of site-specific competitive chemical modification of single cysteine mutants. As a result, it was revealed that MacB is composed of four transmembrane (TM) segments with a cytoplasmic N-terminal nucleotide binding domain of about 270 amino acid residues and a periplasmic large hydrophilic polypeptide between TM segments 1 and 2 of about 200 amino acid residues.     

Analysis of the feed-forward effects of sink activity on the photosynthetic source-sink balance in single-rooted sweet potato leaves. I. Activation of RuBPcase through the development of sinks

Single-rooted sweet potato leaves having a petiole with a fragment of stem allocated exceptionally large amounts of photosynthates to tuberous roots, the only major storage organ, throughout an experimental period of 50 d. The increase in photosynthetic activity for CO(2) fixation depended exclusively on the development of sink activity due to the growth of tuberous roots. Thus this model expressed a remarkable feed-forward effect on the photosynthetic source-sink balance. The level of ribulose-1,5-bisphosphate carboxylase (RuBPcase) protein in the leaves increased continuously during the period. The lowered initial as well as total activity of RuBPcase observed at the start of the experiment was raised with the cancellation of the sink-limited state due to the development of tuberous roots. The maximum activity determined after removing some inhibitor(s) from the enzyme by treating the leaf extract with SO(4)(2-) was much greater than the total activity and remained approximately constant throughout the experimental period. The clear decrease in the difference between maximum and total activities with the development of tuberous roots might reflect the reactivation of RuBPcase due to the removal of some inhibitor(s) from the enzyme through the cancellation of the sink-limited state.     

Synthesis,further biological evaluation and pharmacodynamics of newly discovered inhibitors of TNF-alpha production

(1S,2R)-2-Acylamino-1-methyl-2-phenylethyl phosphate derivatives 2a, 2b, 3a, and 5a, which are conformationally restricted and metabolically stable analogues of (2R)-2-acylamino-2-phenylethyl phosphate derivatives 1a and 1b, are a new class of inhibitors of TNF-alpha production. More efficient alternative synthesis of a key intermediate, (1R,2S)-1-amino-1-(3-methoxyphenyl)propan-2-ol hydrochloride (9), was achieved using one-step, three-component coupling of 3-methoxyphenyl boronic acid (13), (5S)-2,2,5-trimethyl-1,3-dioxolan-4-ol (14), and amino diphenyl methane (15), [as reported in J. Am. Chem. Soc. 1998, 120, 11798]. Evaluation of the hypotensive activity of these compounds was done to assess one of their side effects. Among the compounds tested, the above-mentioned four compounds (2a, 2b, 3a, and 5a) were identified as inhibitors with both sufficient potency and an acceptable safety margin regarding their hypotensive activity. The pharmacodynamics of these compounds were also investigated. Single-dose pharmacokinetic data for compounds 2a, 2b, 3a, and 5a are displayed. These compounds were estimated to be mainly metabolized by the liver in the species tested based on their in vitro stability in tissue homogenates and plasma. A representative compound, 2a, showed good linearity of its plasma concentration after intravenous injection.     

Natural alpha interferon-producing cells respond to human immunodeficiency virus type 1 with alpha interferon production and maturation into dendritic cells

Natural alpha interferon (IFN-alpha)-producing cells (IPCs) are now recognized as identical to plasmacytoid dendritic cell (DC) precursors in human blood and are thought to play an important role in antiviral immunity. In the present study, we examined the susceptibility as well as the cellular responses of IPCs to human immunodeficiency virus type 1 (HIV-1) infection. HLA-DR(+) CD11c(-) lineage-negative cells (IPCs) were purified from peripheral blood mononuclear cells by magnetic-bead separation and cell sorting. We substantiated that IPCs expressing the major HIV-1 coreceptors, CXCR4 and CCR5, are susceptible to infection of both T-cell-line-tropic NL4-3 and macrophage-tropic JR-CSF HIV-1 by quantification of HIV-1 p24 in the culture supernatants and by provirus integration assay using human conserved Alu-HIV-1 long terminal repeat PCR. To evaluate the cellular response of IPCs to HIV-1, we examined IFN-alpha production and their differentiation into DCs. After incubation with either NL4-3 or JR-CSF, IPCs produced a large amount of IFN-alpha and at the same time underwent morphological differentiation into DCs with upregulation of CD80 and CD86. Heat inactivation of the supernatants containing HIV-1 did not affect the IFN-alpha production and maturation, whereas removal of virions by ultracentrifugation completely nullified both biological effects, indicating that these cellular responses do not require actual HIV-1 infection but are elicited by interaction with HIV-1 virions or certain viral components. In conclusion, these data strongly suggest that IPC can directly recognize and respond to HIV-1 with IFN-alpha production, which is crucial for preventing progress of HIV-1 infection and occurrence of opportunistic infection.     

CD117+ stem cells play a key role in therapeutic angiogenesis induced by bone marrow cell implantation

Therapeutic angiogenesis can be induced by the implantation of bone marrow mononuclear cells. We investigated the roles of mature mononuclear cell and stem cell fractions in bone marrow in this treatment. Although CD34 is the most popular marker for stem cell selection for inducing therapeutic angiogenesis, we separated CD117-positive cells (CD117+) from mature bone marrow mononuclear cells [CD117-negative cells (CD117-)] from mice using the antibody to the stem cell receptor, because some of the bone marrow stem cells that express CD117+ and CD34- might generate angiogenic cytokines and differentiate into endothelial cells. The angiogenic potency of CD117+ and CD117- cells was investigated in vitro and in vivo. Significantly higher levels of VEGF were secreted from the CD117+ cells than from the CD117- cells (P < 0.001). Most of the CD117- cells died, but the CD117+ cells grew well and differentiated into endothelial cells within 14 days of culture. The CD117+ cells survived and were incorporated in microvessels within 14 days of being implanted into the ischemic hindlimbs of mice, but the CD117- cells did not. The microvessel density and blood perfusion of the ischemic hindlimbs were significantly higher in the CD117+ cell-implanted mice than in the CD117- cell-implanted mice (P < 0.01). The microvessel density in ischemic hindlimbs was also significantly higher in the CD117+ cell-implanted mice than in the total bone marrow cell-implanted mice (P < 0.05). Thus CD117+ stem cells play a key role in the therapeutic angiogenesis induced by bone marrow cell implantation.     

Highly potent inhibitors of TNF-alpha production. Part 2: identification of drug candidates

Metabolic stabilization of the chemical lead 1, which is a structurally novel inhibitor of TNF-alpha production, was accomplished by introducing a (1S)-methyl group into the optically active backbone. As a result, 2, 3 and 4 were identified as drug candidates and evaluated pharmacologically. The analysis of an active conformer was also carried out.     

Highly potent inhibitors of TNF-alpha production. Part I: discovery of new chemical leads and their structure-activity relationships

Discovery of new chemical leads of inhibitors for TNF-alpha production starting from the chemical modification of 1 is reported. Further biological studies of 1 to disclose the site of its action strongly suggested that 1 inhibits LPS-induced TNF-alpha expression in the liver and spleen of mice. Structure-activity relationships (SARs) are also discussed and full details including the chemistry are reported.     

Release of hepatocyte growth factor from mechanically stretched skeletal muscle satellite cells and role of pH and nitric oxide

Application of mechanical stretch to cultured adult rat muscle satellite cells results in release of hepatocyte growth factor (HGF) and accelerated entry into the cell cycle. Stretch activation of cultured rat muscle satellite cells was observed only when medium pH was between 7.1 and 7.5, even though activation of satellite cells was accelerated by exogenous HGF over a pH range from 6.9 to 7.8. Furthermore, HGF was only released in stretched cultures when the pH of the medium was between 7.1 and 7.4. Conditioned medium from stretched satellite cell cultures stimulated activation of unstretched satellite cells, and the addition of anti-HGF neutralizing antibodies to stretch-conditioned medium inhibited the stretch activation response. Conditioned medium from satellite cells that were stretched in the presence of nitric-oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester hydrochloride did not accelerate activation of unstretched control satellite cells, and HGF was not released into the medium. Conditioned medium from unstretched cells that were treated with a nitric oxide donor, sodium nitroprusside dihydrate, was able to accelerate the activation of satellite cells in vitro, and HGF was found in the conditioned medium. Immunoblot analysis indicated that both neuronal and endothelial NOS isoforms were present in satellite cell cultures. Furthermore, assays of NOS activity in stretched satellite cell cultures demonstrated that NOS is stimulated when satellite cells are stretched in vitro. These experiments indicate that stretch triggers an intracellular cascade of events, including nitric oxide synthesis, which results in HGF release and satellite cell activation.     

KL-6, a human MUC1 mucin, promotes proliferation and survival of lung fibroblasts

The serum level of KL-6, a MUC1 mucin, is a clinically useful marker for various interstitial lung diseases. Previous studies demonstrated that KL-6 promotes chemotaxis of human fibroblasts. However, the pathophysiological role of KL-6 remains poorly understood. Here, we further investigate the functional aspects of KL-6 in proliferation and apoptosis of lung fibroblasts. KL-6 accelerated the proliferation and inhibited the apoptosis of all human lung fibroblasts examined. An anti-KL-6 monoclonal antibody counteracted both of these effects induced by KL-6 on human lung fibroblasts. The pro-fibroproliferative and anti-apoptotic effects of KL-6 are greater than and additive to those of the maximum effective concentrations of platelet-derived growth factor, basic fibroblast growth factor, and transforming growth factor-beta. These findings indicate that increased levels of KL-6 in the epithelial lining fluid may stimulate fibrotic processes in interstitial lung diseases and raise the possibility of applying an anti-KL-6 antibody to treat interstitial lung diseases.