(17/173) Micrococcus luteus teichuronic acids activate human and murine monocytic cells in a CD14- and toll-like receptor 4-dependent manner.
Teichuronic acid (TUA), a component of the cell walls of the gram-positive organism Micrococcus luteus (formerly Micrococcus lysodeikticus), induced inflammatory cytokines in C3H/HeN mice but not in lipopolysaccharide (LPS)-resistant C3H/HeJ mice that have a defect in the Toll-like receptor 4 (TLR4) gene, both in vivo and in vitro, similarly to LPS (T. Monodane, Y. Kawabata, S. Yang, S. Hase, and H. Takada, J. Med. Microbiol. 50:4-12, 2001). In this study, we found that purified TUA (p-TUA) induced tumor necrosis factor alpha (TNF-alpha) in murine monocytic J774.1 cells but not in mutant LR-9 cells expressing membrane CD14 at a lower level than the parent J774.1 cells. The TNF-alpha-inducing activity of p-TUA in J774.1 cells was completely inhibited by anti-mouse CD14 monoclonal antibody (MAb). p-TUA also induced interleukin-8 (IL-8) in human monocytic THP-1 cells differentiated to macrophage-like cells expressing CD14. Anti-human CD14 MAb, anti-human TLR4 MAb, and synthetic lipid A precursor IV(A), an LPS antagonist, almost completely inhibited the IL-8-inducing ability of p-TUA, as well as LPS, in the differentiated THP-1 cells. Reduced p-TUA did not exhibit any activities in J774.1 or THP-1 cells. These findings strongly suggested that M. luteus TUA activates murine and human monocytic cells in a CD14- and TLR4-dependent manner, similar to LPS. (+info)
(18/173) Crystal structure of cis-prenyl chain elongating enzyme, undecaprenyl diphosphate synthase.
Undecaprenyl diphosphate synthase (UPS) catalyzes the cis-prenyl chain elongation onto trans, trans-farnesyl diphosphate (FPP) to produce undecaprenyl diphosphate (UPP), which is indispensable for the biosynthesis of bacterial cell walls. We report here the crystal structure of UPS as the only three-dimensional structure among cis-prenyl chain elongating enzymes. The structure is classified into a protein fold family and is completely different from the so-called "isoprenoid synthase fold" that is believed to be a common structure for the enzymes relating to isoprenoid biosynthesis. Conserved amino acid residues among cis-prenyl chain elongating enzymes are located around a large hydrophobic cleft in the UPS structure. A structural P-loop motif, which frequently appears in the various kinds of phosphate binding site, is found at the entrance of this cleft. The catalytic site is determined on the basis of these structural features, from which a possible reaction mechanism is proposed. (+info)
(19/173) Identification of Significant residues for homoallylic substrate binding of Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase.
The primary structure of cis-prenyltransferase is totally different from those of trans-prenyltransferases (Shimizu, N., Koyama, T., and Ogura, K. (1998) J. Biol. Chem. 272, 19476-19481). To better understand the molecular mechanism of enzymatic cis-prenyl chain elongation, we selected seven charged residues in the conserved Region V and two of Phe-Ser motif in Region III of undecaprenyl diphosphate synthase of Micrococcus luteus B-P 26 for substitutions by site-directed mutagenesis and examined their effects on substrate binding and catalysis. Kinetic studies indicated that replacements of Arg-197 or Arg-203 with Ser, and Glu-216 with Gln resulted in 7-11-fold increases of Km values for isopentenyl diphosphate and 18-1200-fold decreases of kcat values compared with those of the wild-type enzyme. In addition, two mutants with respect to the Phe-Ser motif in Region III, F73A and S74A, showed 16-32-fold larger Km values for isopentenyl diphosphate and 12-16-fold lower kcat values than those of the wild-type. Furthermore, product analysis indicated that three mutants, F73A, S74A, and E216Q, yielded shorter chain prenyl diphosphates as their main products. These facts together with the protein structural analysis recently carried out (Fujihashi, M., Zhang, Y.-W., Higuchi, Y., Li, X.-Y., Koyama, T., and Miki, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4337-4342) indicated that the diphosphate moiety of homoallylic substrate is electrostatically recognized by the three charged amino acids, Arg-197, Arg-203, and Glu-216, in Region V and the Phe-Ser motif in Region III, also indispensable for homoallylic substrate binding as well as catalytic function. It was suggested that the undecaprenyl diphosphate synthase takes a different mode for the binding of isopentenyl diphosphate from that of trans-prenyl chain elongating enzymes. (+info)
(20/173) Optimization of annealing temperature to reduce bias caused by a primer mismatch in multitemplate PCR.
To reduce PCR bias derived from a primer mismatch, the effect of the annealing temperature on the product ratio was investigated by denaturing gradient gel electrophoresis analysis of PCR products from a mixture of perfect-match and one-mismatch templates. These templates were generated by PCR from Pediococcus acidilactici for one mismatch and Micrococcus luteus for the perfect match. PCRs showed that the bias was reduced at lower temperatures. An environmental sample was also examined. (+info)
(21/173) Development and validation of limited-sampling strategies for predicting amoxicillin pharmacokinetic and pharmacodynamic parameters.
Amoxicillin plasma concentrations (n = 1,152) obtained from 48 healthy subjects in two bioequivalence studies were used to develop limited-sampling strategy (LSS) models for estimating the area under the concentration-time curve (AUC), the maximum concentration of drug in plasma (C(max)), and the time interval of concentration above MIC susceptibility breakpoints in plasma (T>MIC). Each subject received 500-mg amoxicillin, as reference and test capsules or suspensions, and plasma concentrations were measured by a validated microbiological assay. Linear regression analysis and a "jack-knife" procedure revealed that three-point LSS models accurately estimated (R(2), 0.92; precision, <5.8%) the AUC from 0 h to infinity (AUC(0-infinity)) of amoxicillin for the four formulations tested. Validation tests indicated that a three-point LSS model (1, 2, and 5 h) developed for the reference capsule formulation predicts the following accurately (R(2), 0.94 to 0.99): (i) the individual AUC(0-infinity) for the test capsule formulation in the same subjects, (ii) the individual AUC(0-infinity) for both reference and test suspensions in 24 other subjects, and (iii) the average AUC(0-infinity) following single oral doses (250 to 1,000 mg) of various amoxicillin formulations in 11 previously published studies. A linear regression equation was derived, using the same sampling time points of the LSS model for the AUC(0-infinity), but using different coefficients and intercept, for estimating C(max). Bioequivalence assessments based on LSS-derived AUC(0-infinity)'s and C(max)'s provided results similar to those obtained using the original values for these parameters. Finally, two-point LSS models (R(2) = 0.86 to 0.95) were developed for T>MICs of 0.25 or 2.0 microg/ml, which are representative of microorganisms susceptible and resistant to amoxicillin. (+info)
(22/173) Structural study of novel antimicrobial peptides, nigrocins, isolated from Rana nigromaculata.
Novel cationic antimicrobial peptides, named nigrocin 1 and 2, were isolated from the skin of Rana nigromaculata and their amino acid sequences were determined. These peptides manifested a broad spectrum of antimicrobial activity against various microorganisms with different specificity. By primary structural analysis, it was revealed that nigrocin 1 has high sequence homology with brevinin 2 but nigrocin 2 has low sequence homology with any other known antimicrobial peptides. To investigate the structure-activity relationship of nigrocin 2, which has a unique primary structure, circular dichroism (CD) and homonuclear nuclear magnetic resonance spectroscopy (NMR) studies were performed. CD investigation revealed that nigrocin 2 adopts mainly an alpha-helical structure in trifluoroethanol (TFE)/H(2)O solution, sodium dodecyl sulfate (SDS) micelles, and dodecylphosphocholine micelles. The solution structures of nigrocin 2 in TFE/H(2)O (1:1, v/v) solution and in SDS micelles were determined by homonuclear NMR. Nigrocin 2 consists of a typical amphipathic alpha-helix spanning residues 3-18 in both 50% TFE solution and SDS micelles. From the structural comparison of nigrocin 2 with other known antimicrobial peptides, nigrocin 2 could be classified into the family of antimicrobial peptides containing a single linear amphipathic alpha-helix that potentially disrupts membrane integrity, which would result in cell death. (+info)
(23/173) Demethylation metabolism of roxithromycin in humans and rats.
AIM: To investigate the demethylated metabolites of roxithromycin (RXM) in humans and rats, and to study the antibiotic activity of these metabolites in vitro. METHODS: The demethylated metabolites of RXM in humans and in rats were identified by liquid chromatography-mass spectrometry (LC-MS), and the in vitro antibiotic activities of them against three standard strains were also studied compared with those of the parent drug and some other metabolites of RXM. RESULTS: O-Demethylation of RXM was one of the main metabolic routes of RXM in humans, whereas N-demethylation metabolism was more predominant in rats. O-Demethyl-RXM appeared to be equally effective with RXM. CONCLUSION: The O-demethyl-RXM was an active metabolite in humans, and there were some species differences in RXM demethylation metabolism between humans and rats. (+info)
(24/173) Cutting edge: the toll pathway is required for resistance to gram-positive bacterial infections in Drosophila.
In Drosophila, the response against various microorganisms involves different recognition and signaling pathways, as well as distinct antimicrobial effectors. On the one hand, the immune deficiency pathway regulates the expression of antimicrobial peptides that are active against Gram-negative bacteria. On the other hand, the Toll pathway is involved in the defense against filamentous fungi and controls the expression of antifungal peptide genes. The gene coding for the only known peptide with high activity against Gram-positive bacteria, Defensin, is regulated by both pathways. So far, survival experiments to Gram-positive bacteria have been performed with Micrococcus luteus and have failed to reveal the involvement of one or the other pathway in host defense against such infections. In this study, we report that the Toll pathway, but not that of immune deficiency, is required for resistance to other Gram-positive bacteria and that this response does not involve Defensin. (+info)