Pyridoxal 5 phosphate

Pyridoxal-5’-phosphate, L-Serine, and palmitoyl coenzyme A were purchased from Sigma-Aldrich (St. Louis, MI, USA). Sphingosine-1-phosphate (S1P) was purchased from Avanti Polar Lipids. DMEM culture media and fetal bovine serum (FBS) were purchased from EuroClone Life Science Division (Milan, Italy). l-[³H(G)]-Serine was purchased from PerkinElmer, Inc. (Wellesley, MA, USA).
Human breast cancer cell line MDA-MB-231, from America Type Culture Collection (ATCC), Rockville, MD, USA, was chosen as the source of serine-palmitoyl transferase (SPT) enzyme. Cells were maintained at 5% CO₂, 95% humidity at 37 °C in DMEM, supplemented with 10% FBS and 1% penicillin/streptomycin solution as antibiotics. Cells were seeded in 150 mm Petri dishes (3 × 106 cells in 24 mL) and left to grow for at least 3 days, until 80–90% confluence was reached, before being collected for microsomal membranes fraction extraction, due to the localization of the studied enzyme at this level.

The L. rhamnosus CA15 (DSM 33960) strain was reactivated by inoculation at 2% into de Man Rogosa and Sharp medium (MRS, Oxoid, Milan, Italy) and incubated at 37 °C overnight. The resulting biomass was subcultured five times in decarboxylase medium broth supplemented with 0.005% of pyridoxal-5′-phosphate (Sigma-Aldrich, St. Louis, MO, USA), and the production of biogenic amines (BA) was stimulated by adding either 0.1% histidine, 0.1% lysine, 0.1% tyrosine, or 0.1% ornithine (Sigma-Aldrich, St. Louis, MO, USA) as described by Bover-Cid and Holzapfel [29 (link)]. Biogenic amine extraction and estimation protocols were performed as described previously [30 (link)]. Specifically, extracted and derivatized samples were separated by HPLC (Shimadzu Nexera XL UHPLC system equipped with a SPD M30A diode array detector) using a 250 mm × 46 mm × 5µm Discovery HS C18 column (Sigma-Aldrich) applying a low-pressure gradient of acetonitrile and water. Unlike the reference paper, the separation temperature was kept constant at 26 °C to allow a better reproducibility of the results. Six-point standard curves were generated and considered suitable if they presented a correlation R2 value of >0.99. The unknown samples were assayed in duplicate, and the results were analyzed as average ± standard deviation.

The following chemicals were purchased from Sigma-Aldrich (St. Louis, MO): methanol and acetonitrile (MS grade), ammonium acetate, ascorbic acid, β-mercaptoethanol, formic acid and standards, 5-adenosyl-methionine, 5-adenosyl-homocysteine, ATP, betaine, choline, cyanocobalamin, cystathionine, cysteine, dihydrofolate, dimethylglycine, dUMP, folic acid, folinic acid, glycine, homocysteine, methionine, methylcobalamine, methyl-tetrahydrofolate, NADPH, pyridoxal 5-phosphate, riboflavin, serine, taurine, tetrahydrofolate, thymidine 5-phosphate and Dulbecco's modified Eagle's medium mixed 1:1 with Ham's F-12 (DMEM/F12). Ultrapure type 1 water was obtained from a Milli Q water system (Merck Millipore, Darmstadt, Germany). Matrigel (growth factor-reduced) was purchased from BD Biosciences (San Jose, CA). Recombinant basic fibroblast growth factor (bFGF) was purchased from Mylteni (San Diego, USA).

Celecoxib, niflumic acid, parecoxib, L-kynurenine (sulfate salt), dimethyl sulfoxide (DMSO), sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, sodium phosphate monobasic, sodium phosphate dibasic, glucose, distilled water, Trizma base, acetic acid, pyridoxal 5′-phosphate, 2-mercaptoethanol, pyruvate, and glutamine were obtained from Sigma-Aldrich. High-performance liquid chromatography (HPLC) reagents were purchased from J.T. Baker Chemicals and from Sigma-Aldrich.

E. coli strains DH5α, BL21(DE3), BL21(DE3)plysS and Rosetta(DE3), the plasmids pETDuet-1 and pET-22b, and the FRETWorks S-tag Assay Kit were purchased from Novagen (Madison, WI). Reagents used in plasmid construction, protein expression, and site-directed mutagenesis were bought from Takara (Dalian, China). The affinity matrix nickel-nitrilotriacetic acid (Ni–NTA) superflow is a product of Qiagen (Chatsworth, CA). The YM-10 membrane was supplied by Amicon (USA). Pyridoxal 5′-phosphate and DL-α,β-diaminopropionate (DL-DAP) were from Sigma (USA). DNA sequencing was performed by Invitrogen (Shanghai, China).