Nessler s reagent

Culture media components such as tryptone extract and Bacto yeast extract were purchased from Difco Laboratories, India. Phenylmethylsulfonyl fluoride (PMSF), isopropyl β-D-1-thiogalactopyranoside (IPTG), acrylamide, bis-acrylamide, Tris–HCl, and L-asparagine were from Amresco, United States. Sodium dodecyl sulfate (SDS), ammonium persulfate (APS), dithiothreitol (DTT), urea, Nessler’s reagent, and trichloroacetic acid (TCA) were from Sigma-Aldrich, United States. 2,2,2-Trifluoroethanol was from SRL, India. Tetramethyl ethylenediamine (TEMED), ethylenediaminetetraacetic acid (EDTA), and bromophenol blue were from Bio-Rad, United States. Coomassie Brilliant Blue R-250 and ampicillin were from USB Corporation, United States. Glacial acetic acid and methanol were from Merck’s EMPARTA^®. Ethanol, n-propanol, and glycerol were of analytical grade and were from Spectrochem, India. DEAE-Sepharose Fast Flow media was purchased from GE Healthcare, United Kingdom. BCA assay kit, SDS-PAGE prestained molecular weight marker, and commercial r-hGH were from Thermo Fisher Scientific, United States. Commercial L-asparaginase 5,000 IU/ml, Bionase^® 5K, was purchased from Zydus Cadila, India. RPMI, horse serum (HS), and fetal bovine serum (FBS) were from Gibco BRL, United States. All the other chemicals were of analytical grade.

Eosin Y, pyrene, PC from egg yolk, aminoguanidine, sodium azide, serotonin, benzylamine, Nessler’s reagent, thiobarbituric acid (TBA), trichloroacetic acid (TCA), penicilamine (PA), tert-butyl hydroperoxide (TBHP) and luminol were purchased from Sigma (Merck Life Science LLC, Moscow, Russian Federation). Bovine serum albumin (BSA) (fraction V) and D-glucose were purchased from Life Science (USA). Folin’s reagent was purchased from Applichem (AppliChem GmbH, Darmstadt, Germany).

Nitrilase activity was determined as described by Piotrowski et al. (2001) (link) with some modifications. Cassava tissue was homogenized in an extraction buffer containing 50 mM Tris-HCl (pH 8.5), 2.0 mM EDTA, 8.0 mM cysteine, 2% (w/v) PVP plus and minus (for plant protein quantification) 0.1% (w/v) BSA. Tuberous greenhouse roots were homogenized in a blender for 5 s × 2 s, while in vitro plant material was homogenized by grinding with liquid nitrogen in a motor and pestle. In all cases, the homogenate was filtered through four layers of cheesecloth and centrifuged for 5 min at 22000 g. Approximately 400 μg of plant protein was used in the subsequent enzyme assay. Enzyme extracts were pre-warmed at 37°C for 2 min before being incubated with substrate (10 mM cyanoalanine in 50 mM Tris-HCl, pH 8.5 and 1.0 mM DTT) for 10 min at 37°C. The total reaction volume was 1.0 mL. The reaction was stopped by adding 100 μL of tricarboxylic acid and centrifuged at 22000 g for 2 min. To 500 μL of the supernatant, 1.0 mL of Nessler’s reagent (Sigma-Aldrich²) was added. The samples were incubated at room temperature for 10 min to allow color development. For blank samples, TCA was added at time 0. Absorbance was read at 480 nm and the amount of ammonia produced was estimated using a standard curve.

Samples from the R1, R2, and R3 compartments were centrifuged (10,000 × g, 10 min) and the supernatants analyzed by HPLC as described earlier (Barroso et al., 2015 (link)). Briefly, samples (20 μL) were injected on a HPLC system (Jasco, Tokyo, Japan) equipped with a UV-975 detector. SCFA were separated using a Rezex ROA Organic Acids column (Phenomenex, Macclesfield, United Kingdom) using 5 mM sulphuric acid as mobile phase. The elution profile was monitored at 210 nm and the identification of the peaks was carried out by comparing the retention times of target peaks with those of the standards: acetic, propionic, butyric, formic, succinic, and lactic acids. Calibration curves of these acids were carried out in the concentration range from 1 to 100 mM. Ammonium was determined using the Nessler’s reagent (Sigma) as previously described (Doo et al., 2017 (link)).

Protein content was used to standardize arginine deiminase (ADS) and urease activity and defined as µmol/min/mg protein (Zheng et al., 2017 (link)). In brief, cells from saliva-derived biofilms were added into a mixture with 50 mM arginine hydrochloride (Sigma-Aldrich Canada, Oakville, Ontario, Canada) and 0.5 mM Tris-maleate buffer (pH 6.0) and then incubated together for 120 min at 37°C to allow ammonia generation. The ammonia production was monitored using Nessler’s reagent (Sigma-Aldrich) based on a standard generated with ammonium sulfate. Simultaneously, protein content was measured using Bradford’s assay and bovine serum albumin was used as standard. Lactic dehydrogenase (LDH) activity was determined using an LDH Activity Assay Kit (Sigma-Aldrich), as per the manufacturer’s guidelines (Zheng et al., 2017 (link)). Further, a deviation between the fluoride group (275 and 1250 ppm NaF) and the control group (0 ppm NaF) for the FR strain and WT was calculated separately. Results were shown as the absolute value of the deviation of enzyme activities compared with corresponding 0 ppm (ΔLDH, ΔADS, and ΔUrease).

Enzyme activities were measured in supragingival, subgingival and saliva samples (collected in the first recruitment, n = 15 from each group). urease and ADS activities were determined as described by Nascimento et al.^{16 (link)}. In brief, ammonia generated from incubation (37 °C, 120 min) of 125 μl oral samples (suspended plaque and saliva samples) and a 500 μl mixture [50 mM urea (Sigma-Aldrich, St. Louis, MO, USA) or L-arginine-HCL (Sigma-Aldrich), 0.5 mM Tris-maleate buffer (pH = 6.0)] were measured by Nessler’s reagent (Sigma-Aldrich) with ammonium sulfate as a standard. Meanwhile, protein content in each sample was determined by Bradford’s Assay with bovine serum albumin as a standard. urease and ADS activities were expressed as μmol ammonia produced per min and were normalized to mg of protein (μmol/min/mg). LDH activities in plaque and saliva samples were measured by the LDH Activity Assay Kit (Sigma-Aldrich) according to the manufacturer’s instructions. One unit of LDH was defined as the amount of the enzyme that catalyzed the conversion of lactate into pyruvate to generate 1.0 μmol of NADH per min. LDH activity was expressed as the amount of enzyme (U) per g of protein (U/g).

Nutrient broth (LENNOX), Nutrient agar medium (SIGMA-ALDRICH), Luria Bertani (LB) broth (LENNOX), McConkey agar (SIGMA-ALDRICH), mannitol salt agar (OXOID), skim milk agar (NEOGEN), 3% KOH, starch (SIGMA-ALDRICH), Gram staining kit (MERCK), bacteriological peptone (OXOID), hydrogen peroxide, Kings B medium (SIGMA), Wattman No. 1 disc, oxidase reagent, phenol, 0.5% picric acid (SIGMA-ALDRICH), Kovacs reagent, 2% Sodium carbonate (MERCK), Nessler’s reagent (SIGMA-ALDRICH), dilute iodine, Lead (III) nitrate (Sigma- Aldrich), cadmium nitrate tetrahydrate (Sigma- Aldrich), chromium (III) nitrate (Sigma- Aldrich). Analytical balance (SARTORIUS GMBM GOTTINGEN, Germany), digital weighing machine (Jeweler Precision Balance Model: DH-V600A)steam sterilizer (autoclave), 37ºC incubator (MMM group Medcenter Enrich tungsten GmbH), 37ºC shaker (Irmeco GmbH, Germany), Laminar flow (ESCO Prod Model; EQU/03-EHC; Serial # 2000–0052), sterile dissecting pins, Sterile distilled water, dissecting box, gloves, dissecting board, sterile bottles, 70% ethanol, 500 ml beakers, micropipette, 250 ml conical flasks, test tubes, bacteriological wire loop, Petri plates, glycerol, glass rod, glass slides, coverslips, spirit lamp, microscope, and toothpicks.