L cysteine

Clostridium perfringens strains F5603, NCTC8533, ATCC3624, and strain 13 were used in this study [8 (link)-10 (link)]. For the growth of these strains, TGY medium (3 % tryptic soy broth [TSB] [BD Bioscience, Tokyo, Japan], 2 % glucose, 1 % yeast extract [BD Bioscience, Tokyo, Japan], and 0.1 % L-cysteine [Wako Pure Chemical, Osaka, Japan]) with or without chloramphenicol (10 μg/ml) (Wako Pure Chemical, Osaka, Japan) was used as previously described [8 (link), 9 (link)]. E. coli HB101 was used as the host for the recombinant plasmids used for DNA sequencing or for constructing recombinant plasmids carrying the putative replication region from C. perfringens plasmids. For the selection of E. coli or C. perfringens transformants, TSB agar containing ampicillin (100 μg/ml) (Wako Pure Chemical, Osaka, Japan) or chloramphenicol (30 μg/ml) and Brain Heart Infusion (BHI) (BD Bioscience, Tokyo, Japan) agar with chloramphenicol (15 μg/ml) were used, respectively.

All methods were performed in accordance with the guidelines and regulations of chemical substance management and approved by the committees of chemical substance management in the Sanyo-Onoda City University, Musashino University and National Institute of Neuroscience, National Center of Neurology and Psychiatry. Na₂S₂ (Dojindo, Kumamoto, Japan), Na₂S₃ (Dojindo), Na₂S (Wako pure chemicals, Osaka, Japan), l-cysteine (Wako), and glutathione (Wako) were dissolved at 0.1 M in ultrapure water. These stock solutions were stored at − 80 °C, and were used within a week. 5,7-Dichlorokynurenic acid (DCKA) was obtained from Tocris Bioscience (Avonmouth, UK). d-Serine and the other chemicals were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Propionic acid (PA), butyric acid (BA), L-glycine (Gly), L-alanine (Ala), L-serine (Ser), L-threonine (Thr), L-cysteine (Cys), L-methionine (Met), L-valine (Val), L-leucine (Leu), L-isoleucine (Ile), L-phenylalanine (Phe), L-tryptophan (Trp), L-aspartic acid (Asp), L-glutamic acid (Glu), L-asparagine (Asn), L-glutamine (Glu), L-histidine (His), L-asparagine monohydrate (Arg), L-tyrosine (Tyr), and L-ascorbic acid were purchased from FUJIFILM Wako (Osaka, Japan). Acetic acid (AA), L-lysine (Lys), and sodium butyrate were purchased from Nacalai Tesque (Kyoto, Japan), and proline (Pro) was purchased from Sigma-Aldrich (St. Louis, MO, USA). All chemicals were of analytical grade.

Lipids, such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), N-(carbonyl-methoxypolyethyleneglycol2000)-1,2-distearoyl-sn-glycero-3 phosphatidyl-ethanolamine (DSPE-PEG2000), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2000-Mal), were purchased from NOF Corporation (Tokyo, Japan). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and L-cysteine were purchased from FUJIFILM Wako Pure Chemical Corporation (Tokyo, Japan). Herceptin^® (trastuzumab) was purchased from Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan). 1,1-dioctadecyl-3,3,3,3-tetramethyl-indocarbocyanine perchlorate (DiI) was purchased from Setareh Biotech LLC (Eugene, OR, USA).

Reduced form of L-glutathione was obtained from KOHJIN Life Sciences (Tokyo, Japan). Glutathione disulfide, γ-L-glutamyl-L-Cysteine, L-cysteinyl-glycine, and L-cystine with ¹³C isotopes at C1 and C1′ (L-Cystine-1,1′-¹³C) were purchased from Sigma-Aldrich (St. Louis, MO, USA). L-Cysteine was obtained from Wako Pure Chemicals (Osaka, Japan), 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ) from Synchem (Altenburg, Germany), and the AccQ. Tag Ultra Derivatization Kit from Waters (Milford, MA, USA). Acetonitrile (HPLC-grade), triethylamine, formic acid, trichloroacetic acid (TCA), 2-mercaptoethanol, and N-ethylmaleimide (NEM) were obtained from Nacalai Tesque (Kyoto, Japan). Amino acid derivatives and reagents for peptide synthesis were purchased from Watanabe Chemical (Hiroshima, Japan).

This study was approved by the Animal Experiment Committee of Kochi Prefectural University (Approval No. 2022-7). Animal experiments were conducted in accordance with the animal experiment regulations of Kochi Prefectural University. The animal laboratory was maintained at a room temperature of 24 ± 2 °C, humidity of 55 ± 3%, and a 12-h light/dark cycle (light period: 7:00 AM to 7:00 PM, dark period: 7:00 PM to 7:00 AM). Mice introduced from the breeders were fed an MF diet (Oriental Yeast Co., Ltd., Tokyo, Japan) and drinking water (tap water) ad libitum and were pre-bred for 7 days. The experimental animals comprised 4-week-old female ICR (Slc: ICR) mice (Japan SLC, Shizuoka, Japan).
Feed ingredients included casein (FEED ONE Co., Ltd. Kanagawa, Japan), l-cysteine (FUJIFILM Wako Pure Chemical Corporation), cornstarch (Marusan, Kochi, Japan), edible oil (vitamin E-free) (Tama Biochemical Co., Ltd., Tokyo, Japan), cellulose powder (Oriental Yeast Co., Ltd., Tokyo, Japan), AIN-93G mineral mixture (Oriental Yeast Co., Ltd., Tokyo, Japan), AIN-93 vitamin with choline deuterite (Oriental Yeast Co., Ltd.), and tertiary butylhydroquinone (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).

Porphyromonas gingivalis W83, ATCC 33277, KDP131 (ΔrgpA), KDP132 (ΔrgpB), KDP129 (Δkgp), KDP133 (ΔrgpA ΔrgpB) and KDP 136 (Δkgp ΔrgpA ΔrgpB) [34 (link)] were cultured anaerobically (10% CO₂, 10% H₂, and 80% N₂) at 37°C for 60 h in enriched tryptic soy broth containing 1% tryptone (Difco, Detroit, MI), 3% tryptic soy (Becton Dickinson Co., Franklin Lakes, NJ), 2.5% yeast extract (Becton Dickinson), 0.1% _L-cysteine (Wako Pure Chemical Industries, Osaka, Japan), 5 μg/mL of hemin, and 5 μg/mL of menadione. After three washes with PBS, whole bacterial cells at the log-phase of growth were resuspended in PBS, lyophilized by centrifugation in vacuo and then resuspended in distilled water. Viability monitored by counting colony-forming units (CFU) in blood tryptic soy agar, was 4 × 10⁴ CFU/50 μg of lyophilized whole bacterial cells. Enzyme activities in these cells were blocked by a 15-min incubation with the Rgps inhibitor FPR-cmk and/or the Kgp inhibitor KYT-36 at room temperature (RT) before using the bacterial cells to stimulate human gingival epithelial Ca9-22 cells.