Takashi Takagi

Tohoku University, Sendai, Kagoshima, Japan

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Publications (4)8.1 Total impact

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    ABSTRACT: We previously reported the generation of lipopolysaccharide (LPS)-binding peptides by phage display and chemical modification. Among them, a dodecapeptide designated Li5-025 (K′YSSSISSIRAC′; K′ and C′ denote D-lysine and D-cysteine, respectively) showed a high binding affinity for LPS and was resistant to protease digestion (Suzuki et al., 2010). In the current study, Li5-025-bound silica beads, hereafter referred to as P-beads, were generated and found to be devoid of LPS-neutralizing activity. Thus, LPS bound to the P-beads could be directly used in the Limulus amebocyte lysate (LAL) assay. P-beads bound LPS dissolved in solutions of ethanol, pH 4, pH 10, and 0.5 M NaCl and LPS bound to the P-beads was quantitatively assayed. The sensitivity of this assay was observed to be approximately 0.1 pg/mL LPS. P-beads bound LPS dissolved in antithrombin III (AT III) solution which is a strong inhibitor of activated factor C and B as well as the clotting enzyme in the LAL assay; the inhibitory effect of AT III was completely reversed upon washing the P-beads with 25% acetonitrile. This was employed as the first step for the detection of free LPS in plasma using the LAL assay. LPS added to human plasma at 0 °C followed by application to the P-beads and subsequent washing with 25% acetonitrile resulted in low LPS activity as detected by the LAL assay. However, further washing of the P-beads with 0.1% Triton X100 in 25% acetonitrile resulted high LPS activity. This is the first instance of quantitative detection of free LPS in plasma using the LAL assay, the sensitivity of this method was observed to be 1 pg/mL of LPS. The proteins eluted in the 0.1% Triton X-100 wash were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Two protein bands of 28 kDa and 18 kDa were predominantly observed. Mass spectrometry analysis revealed that the 28 kDa and 18 kDa bands corresponded to apolipoprotein A-I (apoA-I) and apolipoprotein A-II (apoA-II), respectively. ApoA-I and A-II are components of high density lipoprotein (HDL). Thus, it is likely that the P-beads-bound LPS were sequestered by HDL, resulting in neutralization of its toxicity. By this study showed using P-beads, free LPS in plasma can be quantitatively measured by the LAL assay at a consideration of 1 pg/ mL.
    Journal of microbiological methods 05/2014; 100(1). DOI:10.1016/j.mimet.2014.02.018 · 2.03 Impact Factor
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    ABSTRACT: Lipopolysaccharide (LPS), a major constituent of the outer membrane of Gram-negative bacteria, is highly toxic and can cause sepsis or septic shock. Therefore, detection of LPS and the ability to neutralize its toxicity is important. We previously obtained a strong LPS-binding peptide, Li5-001, using the phage display method (Matsumoto et al., 2010. J. Microbiol. Methods. 82, 54-58). We modified the sequence the amino acid sequence of this peptide (KNYSSSISSIHAC), by replacing and deleting amino acids to obtain higher LPS-binding affinity and greater resistance to protease digestion. Consequently we obtained a dodecapeptide, Li5-025 (K'YSSSISSIRAC', K' and C' are D-forms of K and C, respectively) which showed a high affinity for LPS, approximately 1000 folds higher affinity than Li5-001 and Kd value of 0.01 nM. By replacing both N- and C-terminal amino acids from L-type to D-type, the peptide was rendered resistant to protease digestion without altering its overall binding capacity.
    Journal of microbiological methods 11/2010; 83(2):153-5. DOI:10.1016/j.mimet.2010.08.009 · 2.03 Impact Factor
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    Journal of Microbiological Methods 10/2010; 83(1):87. DOI:10.1016/j.mimet.2010.08.002 · 2.03 Impact Factor
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    ABSTRACT: Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. It has strong toxicity and might cause sepsis or septic shock. Thus early detection of LPS and neutralization of LPS toxicity are required. We obtained several new LPS-binding peptides using a phage display method. We synthesized 3 of these peptides and analyzed their binding affinity and capacity to LPS. One of these peptides, named Li5-001, showed high binding affinity to LPS and lipid A; the K(d) values were 10 and 1 nM, respectively. Li5-001 showed a high binding capacity to LPS, and was estimated to bind 130 ng LPS/mg, which is higher than that of polymyxin B (80 ng LPS/mg); however, its LPS-neutralizing activity was low. Li5-001 coupled with beads will be useful for eliminating endotoxin contamination from pharmaceuticals. Its low LPS-neutralizing activity allows to be used in the Limulus amebocyte lysate test without eluting LPS from the Li5-001 coupled beads.
    Journal of microbiological methods 07/2010; 82(1):54-8. DOI:10.1016/j.mimet.2010.04.002 · 2.03 Impact Factor