Kyt 36

Manufactured by Peptide Institute

Sourced in Japan

KYT-36 is a laboratory instrument designed for the detection and quantification of peptides. It utilizes advanced chromatographic techniques to separate and analyze peptide samples with high precision and accuracy. The core function of KYT-36 is to provide researchers with reliable and reproducible data for their peptide-related studies and experiments.

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Lab products found in correlation

Kyt 1, by Peptide Institute (8 mentions) L bapna, by Merck Group (2 mentions) Lps pg ultrapure, by InvivoGen (1 mentions) Tos gpk pna, by Merck Group (1 mentions) Bhi broth, by BD (1 mentions) Millicell cell culture insert, by Merck Group (1 mentions) Standard pg lps, by InvivoGen (1 mentions) Ca 074me, by Peptide Institute (1 mentions) Tak 242, by MedChemExpress (1 mentions) U73122, by Merck Group (1 mentions) Gf109203x, by Merck Group (1 mentions) Ammonium pyrrolidinedithiocarbamate pdtc, by Merck Group (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)

9 protocols using kyt 36

Macrophage Infection Dynamics with P. gingivalis

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Macrophages were infected with viable P. gingivalis, heat-killed P. gingivalis (HK-Pg), OMVs or heat-inactivated OMVs (HI-OMVs) at a multiplicity of infection (MOI) of 10:1, 25:1 or 100:1 bacilli or OMV/cell for 2 h. After 2 h the extracellular bacteria/OMVs were removed and the macrophages were washed with PBS and then treated with fresh media (containing 100 U/ml penicillin, 100 μg/ml streptomycin to kill any remaining extracellular bacteria), and M-CSF (2,000 U/ml) for 24 h. This stimulation protocol was used throughout the study and based on our initial dose-response experiments (see Supplementary Figures 2A,B) and other studies (Taxman et al., 2012 (link); Cecil et al., 2016 (link); Waller et al., 2016 (link)) an MOI of 25:1 was chosen as the optimal dose for this study. For reference, OMVs (at an MOI of 25:1) was equal to a protein concentration of 3.0 μg/ml. In certain experiments cells were stimulated with P. gingivalis-derived LPS (Invivogen; LPS-PG Ultrapure at 100 ng/ml) for 24 h as indicated. These infection protocols were used throughout the study. In specific experiments the competitive gingipain inhibitors, KYT-1 and KYT-36 (Peptide Institute, Osaka, Japan) (Kadowaki et al., 2004 (link)) were included in the media (at 10 μM).

Fleetwood A.J., Lee M.K., Singleton W., Achuthan A., Lee M.C., O'Brien-Simpson N.M., Cook A.D., Murphy A.J., Dashper S.G., Reynolds E.C, & Hamilton J.A. (2017). Metabolic Remodeling, Inflammasome Activation, and Pyroptosis in Macrophages Stimulated by Porphyromonas gingivalis and Its Outer Membrane Vesicles. Frontiers in Cellular and Infection Microbiology, 7, 351.

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Gingipain and PAI-1 Inhibitor Characterization

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Gingipain inhibitors KYT-1 and KYT-36 were obtained from the Peptide Institute (Osaka, Japan). KYT-1 is an Rgp-specific inhibitor and KYT-36 is a Kgp-specific inhibitor [24 (link)]. The PAI-1 antagonist PAI-039 tiplaxtinin was obtained from MedChemExpress (Monmouth Junction, NJ, USA). Recombinant human receptor-associated protein (RAP) and recombinant human PAI-1 (rhPAI-1) mutant were obtained from Sigma-Aldrich (St. Louis, MO, USA). RAP is an antagonist of the low-density lipoprotein receptor-related protein (LRP). It is known that wild-type PAI-1 is not stable and gets converted into an inactive latent form, with a half-life ranging between 1 and 2 h [25 (link)]. The rhPAI-1 mutant is an altered form of human PAI-1 containing 4 mutated amino acids that markedly improved functional stability, because of its inability to convert to the latent form. The residue numbers for the four mutations present in rhPAI-1 mutant are N150H, K154T, Q319L, and M354I [25 (link)]. As a consequence, the mutant protein exhibits markedly increased thermal stability of the active form relative to active wild-type PAI-1. All other reagents were purchased from Sigma-Aldrich unless otherwise indicated.

Song L.T., Tada H., Nishioka T., Nemoto E., Imamura T., Potempa J., Li C.Y., Matsushita K, & Sugawara S. (2021). Porphyromonas gingivalis Gingipains-Mediated Degradation of Plasminogen Activator Inhibitor-1 Leads to Delayed Wound Healing Responses in Human Endothelial Cells. Journal of Innate Immunity, 14(4), 306-319.

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Purification and Activation of Gingipains

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Arg-X gingipains (RgpA and RgpB) and the Lys-X gingipain (Kgp) were purified from spent growth media of P. gingivalis HG66, as described previously [76 (link), 77 (link)]. The concentrations of active Rgp and Kgp gingipains were determined by active site titration using the gingipain-specific inhibitors Kyt-1 and Kyt-36, respectively (Peptide Institute, Japan) [32 (link)]. The purified enzymes were activated by 15 min incubation at 37°C in 100 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl₂, and 20 mM cysteine (pH 7.5), and then diluted to the required concentrations in culture medium supplemented with 10 mM cysteine. Gingipain activity was inhibited by incubating cells with Kyt-1 and/or Kyt-36 (1 μM) for 15 min at 37°C. The efficiency of enzyme inhibition was verified using L-BApNA (Sigma-Aldrich) as a substrate for Arg-X gingipains and Tos-GPK-pNA (Sigma-Aldrich) for the Lys-X gingipain.

Bryzek D., Ciaston I., Dobosz E., Gasiorek A., Makarska A., Sarna M., Eick S., Puklo M., Lech M., Potempa B., Potempa J, & Koziel J. (2019). Triggering NETosis via protease-activated receptor (PAR)-2 signaling as a mechanism of hijacking neutrophils function for pathogen benefits. PLoS Pathogens, 15(5), e1007773.

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Gingipain Inhibitors in P. gingivalis

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Besides using gingipain mutants from two wild-type strains, the influence of gingipain activity was also studied by using gingipain inhibitors. KYT-1 (an arginine gingipain inhibitor) and KYT-36 (a lysine gingipain inhibitor) peptides (15 (link)) (Peptide Institute, Osaka, Japan) were added in the assay to viable P. gingivalis W50 at a concentration of 1×10⁻⁶ mol/ml.

Laheij A.M., van Loveren C., Deng D, & de Soet J.J. (2015). The impact of virulence factors of Porphyromonas gingivalis on wound healing in vitro. Journal of Oral Microbiology, 7, 10.3402/jom.v7.27543.

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Cultivation and Manipulation of P. gingivalis

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Porphyromonas gingivalis wild-type strain ATCC 33277 was grown on blood agar plates as described elsewhere (24 (link)). After anaerobic culture for 5–7 days at 37°C, bacteria were inoculated into brain–heart infusion (BHI) broth (Becton Dickinson) supplemented with 0.5 mg/ml L-cysteine, 10 μg/ml hemin and 0.5 μg/ml vitamin K, and cultured o/n in an anaerobic chamber (85% N₂, 10% CO₂, and 5% H₂). Bacteria were then washed in PBS, resuspended in fresh BHI broth at optical density (OD)_600nm = 0.1 and cultured for ~20 h. A bacterial suspension at OD_600nm = 1 [corresponding to 10⁹ colony-forming units (CFU)/ml] in PBS was prepared and used for experiments. In some experiments, bacteria were heat-inactivated by 30 min incubation at 60°C or were treated with specific gingipain inhibitors KYT-1 and KYT-36 (Peptide Institute Inc.). 1μM KYT-1 and KYT-36 were incubated with bacteria for 20 min at 37°C prior to infection and were added to culture media for the duration of infection.

Maksylewicz A., Bysiek A., Lagosz K.B., Macina J.M., Kantorowicz M., Bereta G., Sochalska M., Gawron K., Chomyszyn-Gajewska M., Potempa J, & Grabiec A.M. (2019). BET Bromodomain Inhibitors Suppress Inflammatory Activation of Gingival Fibroblasts and Epithelial Cells From Periodontitis Patients. Frontiers in Immunology, 10, 933.

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Purification and Activation of Gingipains

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Gingipains (RgpA, RgpB, Kgp) were purified from spent growth medium of P. gingivalis HG66 as described previously (47 (link), 48 (link)). Active-site titration with specific inhibitors KYT-1 (for RgpA/B) and KYT-36 (for Kgp) (Peptide Institute, Inc.) was performed using l-BApNa and N-(p-tosyl)-Gly-Pro-Lys-4-nitroanilide, respectively (Sigma-Aldrich), to determine the concertation of active gingipains (49 (link)). Substrate hydrolysis was measured at 405 nm for 40 min, and activity was presented as milli-optical density units per minute per microliter of sample. Before stimulation of eucaryotic cells, enzymes were activated with 10 mM l-cysteine as described recently (11 (link)).

Ciaston I., Budziaszek J., Satala D., Potempa B., Fuchs A., Rapala-Kozik M., Mizgalska D., Dobosz E., Lamont R.J., Potempa J, & Koziel J. (2022). Proteolytic Activity-Independent Activation of the Immune Response by Gingipains from Porphyromonas gingivalis. mBio, 13(3), e03787-21.

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Endothelial Monolayer Permeability Assay

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Endothelial monolayer permeability was measured using a transwell assay system. Millicell^® cell culture inserts (Merk Millipore, Watford, UK) were fibronectin coated (10 μg/mL) prior to seeding with HDMEC that were cultured until confluent. Pg W83 were treated with KYT-1 or KYT-36 (2 μM; Peptides International, Louisville, USA) to inhibit gingipain activity as previously described.[28 (link)] HDMEC cells were infected in the absence or presence of Pg W83 (with or without inhibitors) at a MOI of 1:1000 for 1.5 h at 37°C in non-supplemented MV medium. Bacteria were removed, inserts transferred to a new 12-well plate with 500 μL supplemented MV medium, and 450 μL of MV supplemented medium added to the apical compartment of the insert together with 70 kDa fluorescent labelled dextran, a molecular weight that does not readily pass through a confluent endothelial monolayer (ThermoFisher Scientific, Loughborough, UK; final concentration of 65 μg/mL). Dextran leakage through the cell monolayer from apical compartment of the insert to the bottom well was monitored for up to 5 h by measuring the fluorescence intensity (excitation 494 nm, emission 521 nm; TECAN Ltd, Männedorf, Switzerland) of 250 μL medium aspirated from the bottom well; 250 μL of fresh medium was added to the bottom well for further readings. Inserts without cells were used as control wells.

Farrugia C., Stafford G.P., Potempa J., Wilkinson R.N., Chen Y., Murdoch C, & Widziolek M. (2020). Mechanisms of vascular damage by systemic dissemination of the oral pathogen Porphyromonas gingivalis. The FEBS journal, 288(5), 1479-1495.

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Peptide Reagents for Inflammation

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hBD3, CA-074Me, KYT-1, and KYT-36 were purchased from the Peptide Institute Inc. (Osaka, Japan), and Z-Arg-Leu-Arg-α-aza-glycil-Ile-Val-OMe (ZRLR) was synthesized by Peptide Institute Inc. Standard Pg LPS was purchased from InvivoGen (San Diego, CA, USA). TAK-242 and C29 were purchased from MedChem Express (Monmouth Junction, NJ, USA). MCC950 was purchased from Adipogen Life Sciences, Inc. (San Diego, CA, USA). Wedelolactone was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).

Inoue E., Minatozaki S., Shimizu S., Miyamoto S., Jo M., Ni J., Tozaki-Saitoh H., Oda K., Nonaka S, & Nakanishi H. (2024). Human β-Defensin 3 Inhibition of P. gingivalis LPS-Induced IL-1β Production by BV-2 Microglia through Suppression of Cathepsins B and L. Cells, 13(3), 283.

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Porphyromonas gingivalis Fimbriae and PGTP2-RL Protocols

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Porphyromonas gingivalis fimbriae and PGTP2-RL were a gift from Dr. T. Ogawa (Asahi University). The properties of P. gingivalis fimbriae [30 (link)–31 (link)] and PGTP2-RL [32 (link)] have been described in detail. The fimbriae were purified from the P. gingivalis 381 (type I fim A) strain prepared by Dr. Ogawa, and LPS containing heterogeneous lipid A structures including tetra- and penta-acylated monophosphorylated lipid A from P. gingivalis prepared as described by Darveau et al. (2004) [33 (link)] was purchased from InvivoGen (San Diego, CA). SB203580, cycloheximide and cytochalasin D were obtained from Calbiochem-Novabiochem Co. (La Jolla, CA). U-73122, GF109203X, ammonium pyrrolidine dithiocarbamate (PDTC) and dimethyl sulfoxide (DMSO) were sourced from Sigma-Aldrich (St. Louis, MO). Phe-Pro-Arg-chloromethyl ketone (FPR-cmk) was obtained from Enzo Life Sciences (Farmingdale, NY) and KYT-36 was sourced from the Peptide Institute (Osaka, Japan).

Tada H., Matsuyama T., Nishioka T., Hagiwara M., Kiyoura Y., Shimauchi H, & Matsushita K. (2016). Porphyromonas gingivalis Gingipain-Dependently Enhances IL-33 Production in Human Gingival Epithelial Cells. PLoS ONE, 11(4), e0152794.

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