Glycine

Chemical reagents such as Tris, glycine, CuCl₂·2H₂O, KCl, Na₂HPO₄·12H₂O, NaCl, KH₂PO₄, EDTA·2Na and IPTG were purchased from Wako Pure Chemical Industries Ltd., Osaka, Japan (Guaranteed Reagent). The synthetic substrate peptidyl-pNA, Arg-pNA, was purchased from Peptides Institute Inc., Osaka, Japan. Tryptone and yeast extract were purchased from Becton–Dickinson and Company, NJ, USA. A commercially available recombinant PD-1 molecule was used (ENZO Life sciences Inc., Product Number; ENZ-PRT190; PD-1 (aa 25-167) containing a 5′-His-tag, V5 epitope tag spacer, and FLAG-tag.

Schizont-rich whole blood was obtained from P. yoelii infected mouse tail and prepared air-dried thin smears on glass slides. The smears were fixed in 4% paraformaldehyde containing 0.0075% glutaraldehyde (Nacalai Tesque) in PBS at room temperature (RT) for 15 min, rinsed with 50 mM glycine (Wako) in PBS. Samples were permeabilized with 0.1% Triton X–100 (Calbiochem) in PBS for 10 min, then blocked with 3% BSA (Sigma) in PBS at RT for 30 min. Next, samples were immunostained with primary antibodies using mouse anti–PyEBL [26 (link)] (final 1:500) and Rabbit anti–PyAMA1 [45 (link)] (a gift from Takafumi Tsuboi, final concentration 1:500) at 37°C for 1 h. This was followed by 3 washes with PBS then incubation with Alexa Fluor 488 goat anti–mouse and Alexa Fluor 594 goat anti–rabbit antibodies (Invitrogen; final 1:1000) in 3% BSA in PBS at 37°C for 30 min. Parasite nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI; Invitrogen, final 0.2 μg/mL). Stained parasites were mounted with Prolong Gold antifade reagent (Invitrogen). Slides were visualized using a fluorescence microscope (Axio imager Z2; Carl Zeiss) with 100x oil objective lens (NA 1.4, Carl Zeiss). Images were captured using a CCD camera (AxioCam MRm; Carl Zeiss) and imaged using AxioVision software (Carl Zeiss). Mann-Whitney U tests were performed using Graphpad Prism software (v6.01).

All of the amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, ornithine, and citrulline), pyroglutamic acid, urocanic acid, urea, and lactic acid were purchased from Wako (Osaka, Japan). All of the SIL-IS were purchased from JUNSEI (Tokyo, Japan). Deionized water was produced in-house using a Milli-Q gradient water purification system (Merck Millipore, Darmstadt, Germany). The SIL-IS mixture solution containing all of the SIL compounds was prepared in deionized water with each having a final concentration of 0.5 μmol/mL. The NMF mixture solutions containing all of the amino acids and pyroglutamic acid, urocanic acid, urea, lactic acid were prepared in deionized water with a final concentration ranging between 0.025–12.5 μmol/mL for each compound.

One hundred milliliters of sample was collected from each site in sterile bottles and centrifuged at 3,000 × g for 30 min. The deposits were resuspended in 1 mL phosphate-buffered saline (PBS) as concentrates. Concentrated samples were heated at 50°C for 30 min and spread onto the surface of GVPC. GVPC is BCYE supplemented with glycine (Wako, Osaka, Japan, 3 mg/mL), vancomycin HCl (Wako, 1 μg/mL), polymyxin B (Wako, 80 IU/mL), and sulfate cycloheximide (Wako, 80 μg/mL). Plates were incubated at 37°C and inspected daily. Isolated bacteria were grown on BCYE or BCYE without cysteine at 37°C. Cysteine auxotrophic bacteria were used for PCR analysis.

Modified mixtures of DMEM were prepared by adding selected components from the 14 amino acids and the eight vitamins contained in basic DMEM into HBSS (pH 7). The concentration of each component is as follows: Amino acids: 0.084 g/L L-Arginine HCl (Tokyo Chemical Industry Co., Ltd. (TCI), Japan), 0.0626 g/L L-Cystine diHCl (TCI), 0.03 g/L Glycine (FUJIFILM Wako Pure Chemical Corporation. (Wako), Japan) 0.042 g/L L-Histidine HCl monohydrate (TCI), 0.105 g/L L-Isoleucine (Wako), 0.105 g/L L-Leucine (TCI), 0.146 g/L L-Lysine monoHCl (TCI), 0.03 g/L Methionine (TCI), 0.066 g/L L-Phenylalanine (TCI), 0.042 g/L L-Serine (TCI), 0.095 g/L L-Threonine (TCI), 0.016 g/L L-Tryptophan (Sigma), 0.12037 g/L L-Tyrosine Disodium Salt Dihydrate (NACALAI TESQUE, INC., Japan), 0.094 g/L L-Valine (TCI). Vitamins include 0.004 g/L Choline Cl (TCI), 0.004 g/L Folic acid (Wako), 0.0072 g/L myo-Inositol (TCI), 0.004 g/L Nicotinamide (TCI), 0.004 g/L Calcium D-pantothenate (TCI), 0.00404 g/L Pyridixine HCl (Wako), 0.0004 g/L Riboflavin (Wako), and 0.004 g/L Thiamine HCl hydrate (TCI).

Drugs were dissolved in Krebs solution and applied by perfusion via a three-way stopcock without any change in either the perfusion rate or the temperature. The drugs used in this study were Ultiva®, 2 mg (Janssen Pharmaceutical K.K., Tokyo, Japan), TTX (Wako, Osaka, Japan), glycine (Wako) and naloxone hydrochloride (Wako). All drugs were diluted to their final concentration in Krebs solution immediately before use. A 2-mg Ultiva® vial contains 15 mg glycine as an adjunct. Ultiva® containing 50 μM remifentanil hydrochloride with 2 mM glycine was superfused on to spinal cord slice preparations in vivo and ex vivo. The plasma concentration of remifentanil achieved by an intravenous infusion of Ultiva® at 0.05–0.20 μg·kg⁻¹ min⁻¹ is approximately 1–5 ng/ml [14 (link), 15 (link)]. Remifentanil has a rapid blood-brain equilibration half-time because of its rapid onset of action. Although the transit of remifentanil to the cerebrospinal fluid is not completely understood, the concentration of Ultiva® superfused was not determined from the plasma concentration, but from the effect obtained in a previous study of Ultiva® or pure remifentanil [5 (link), 16 (link)].