Impacts specific to benthic communities at SMS deposits were reviewed by Van Dover, 2007 and Van Dover, 2011, and are summarized in Table 3. Alongside the obviously negative impacts of mining, such as the loss of sulphide habitat and biodiversity, the search for commercially viable deposits and the environmental surveys carried out by or for mining companies, will have benefits for science (reviewed by Van Dover, 2007 and Van Dover, 2011). The discovery GDC-0068 clinical trial of new SMS sites will occur at a faster pace, and there will be an improved understanding of SMS deposit ecology through the involvement of scientists in impact assessment studies and long-term monitoring

programs. Through industry-led scientific programs, new species could be discovered and the knowledge of life in extreme environments will expand. The management of SMS mining is controlled by different PLX-4720 clinical trial legislation according to the jurisdiction under which the proposed mining project falls. Within the EEZ or legal continental shelf of a country, all mining regulation and management falls under national jurisdiction. All seabed that does not fall within the EEZ or legal continental shelf of a country is termed

‘the Area’ and is managed by the International Seabed Authority (ISA) as determined by the 1982 United Nations Convention on the Law of the Sea. All States party to the Convention must apply to the ISA for licences to prospect, explore and exploit mineral resources in the Area. The ISA has issued regulations governing prospecting and exploration for SMS deposits, which were adopted in May 2010 (International

Seabed Authority, 2010). Contractors must establish environmental baselines against which impacts from mining activities can be assessed, carry out environmental monitoring programmes, and take measures to prevent, reduce, and control pollution and other hazards to the marine environment (see Sections 6 and 7). Contractors must assess if serious harmful effects to vulnerable marine ecosystems, such as those associated with hydrothermal vents, will occur as a results Bacterial neuraminidase of mining activity, and applications for mining can be rejected where substantial evidence indicates the risk of serious harm to the marine environment. Other international conventions, such as the Stockholm Declaration (1972) (http://www.unep.org/Documents), the Rio Declaration (1992) (http://www.unep.org/Documents), the Convention on Biodiversity (1993) (http://www.cbd.int/convention/text/) and the World Summit on Sustainable Development (2002) (http://www.un.org/jsummit/html/documents/summit_docs.html), influence the drafting of marine mining legislation by signatory countries. The Stockholm and Rio Declarations emphasise the need for environmental protection and environmental impact assessment in sustainable development, alongside the need to share scientific knowledge and adopt the ‘precautionary principle’.

In addition, endogenous PGs are also necessary for normal bone repair [20] and a critical role for COX-2 and PGE2 in triggering Wnt/β-catenin signaling in the anabolic response to mechanical loading has been proposed [21]. Four G-protein coupled receptors, EP1, EP2, EP3 and EP4, are associated with effects of PGE2. EP2 and EP4, which activate Gαs and stimulate cAMP formation, have predominant roles in both PGE2-stimulated bone resorption and formation [15]. EP3 is coupled to Gαi and inhibits cAMP, while EP1 acts largely by increasing calcium flux and perhaps protein kinase C (PKC) [22]. Because PTH induces PGE2 production

and because PTH and PGE2 both have major actions via similar Gαs/cAMP-activated pathways [23] and [24], our initial hypothesis was that IDH mutation PGE2 was the local mediator of some of the anabolic actions of PTH. However, we found intermittent PTH in vivo to be more anabolic in Cox-2 KO mice than in WT mice, suggesting an inhibitory interaction of PTH and PGs [25]. In the current study, we extend our initial findings on the inhibitory interaction of PTH and PGs in vitro [26] to show that the stimulatory effect of PTH on OB differentiation in BMSCs occurred only when COX-2 activity was absent in both mesenchymal and hematopoietic

cells. Using co-cultures and conditioned media (CM) from bone marrow macrophages (BMMs), we show that the inhibition of PTH-stimulated OB differentiation Crizotinib research buy was mediated by a factor or factors secreted by hematopoietic cells committed to the OC lineage in response to COX-2 produced PGs or to added PGE2. This study reveals a new role for COX-2 and PGE2 in regulating PTH-stimulated responses in bone and a new example of regulation of OB differentiation by OCs. PGE2, NS398, MRE-269 (prostaglandin IP receptor agonist), dinoprost (PGF2α

receptor agonist) and all other prostanoids used were from Cayman Chemical Company (Ann Arbor, MI). Recombinant either mouse macrophage-colony stimulating factor (M-CSF), osteoprotegerin (OPG)/Fc-chimera and RANKL were from R&D systems (Minneapolis, MN). Bovine PTH (bPTH; 1–34) and all other chemicals were from Sigma (St. Louis, MO), unless otherwise noted. Mice with disruption of Ptgs2, which produce no functional COX-2 protein, called Cox-2 knockout (KO) mice, in a C57BL/6, 129SV background were the gift of Scott Morham [27]. Ptger2 and Ptger4 KO mice in C57BL/6, 129 backgrounds were gifts from Richard and Matthew Breyer [28] and [29]. All KO mice were backcrossed more than 16 generations into the CD-1 (outbred) background. Breeding colonies were refreshed twice a year by regenerating maintenance colonies from mice heterozygous for the deleted or disrupted gene mated with WT mice from Jackson Laboratory (Bar Harbor, ME). For experiments, Cox-2 KO mice were bred by KO × KO mating, and Ptger2 and Ptger4 KO mice were bred by heterozygous × heterozygous mating.

, 2010). Our study demonstrated PF-02341066 manufacturer that the Na+/K+-ATPase activity was not modified by IBTC at any of the concentrations tested, indicating that IBTC

has no toxic properties to neurons. Previous reports have demonstrated the importance of thiol groups for Na+/K+-ATPase catalysis and –SH groups of this enzyme are highly susceptible to oxidizing agents (Bavaresco et al., 2003 and de Assis et al., 2003). The unchanged NPSH levels found here are in agreement with the unchanged activities of ALA-D and Na+/K+-ATPase. We also demonstrate that IBTC did not alter the activity of AChE and BChE, enzymes related to dysfunctions in the cholinergic selleck products neurotransmission (Mukherjee et al., 2007) and

to systemic inflammatory conditions, such as diabetes mellitus, hypertension, insulin resistance, and hyperlipidemia (Das, 2007). AChE and BChE are strongly related to the intoxications caused by pesticides and the implications of pesticides residues on human health have yet to be comprehensively documented. Pesticides may induce oxidative stress, leading to generation of free radicals and alterations in antioxidants, oxygen free radicals, scavenging enzyme systems, and lipid peroxidation. This way, after verifying that IBTC does not alter antioxidant systems and has no toxic effects, we tested the capacity of IBTC to protect and reactivate the activity of AChE and BChE after inhibition

with MAP. In human erythrocyte ghost and in human plasma BChE, IBTC was able to protect and reactivate both enzymes from MAP inhibition at all concentrations tested (Fig. 5 and Fig. 6). The protective activity for AChE and BChE against MAP inhibition works via competitive inhibition. Molecular docking results indicate that IBTC can enter the active site of AChE by binding to the peripheral anionic site (Trp134 and Tyr124) and Lepirudin internal anionic site (Thr83 and Tyr337), thus preventing MAP from accessing the catalytic residue Ser203 and protecting AChE and BChE from inhibition, and proving that IBTC cannot itself inhibit AChE or BChE, since in vitro tests demonstrate that the presence of IBTC on these sites do not affect AChE and BChE activities. Our most interesting result is that IBTC can reactivate AChE and BChE after inhibition by MAP. As far as we know, there are few compounds that are not oximes that can reactivate AChE and BChE inhibited by OPs and there is no literature concerning the use of thiosemicarbazones against OP intoxication. In that way, our study demonstrates for the first time that a thiosemicarbazone derivate can protect and reactivate AChE and BChE from OP inhibition.

This value is based on internal experience and experiments to distinguish native and punched human skin samples. A lab-specific limit value Crenolanib nmr is necessary due to limited transferability: The measured resistance is dependent on the device, applied frequency, resulting current, ionic strength of the solution as well as the surface area of the skin sample (Fasano et al., 2002). The transepidermal water loss was measured after minimal 1 h of equilibration and drying of the skin surface. The moisture on the skin surface originating from rehydration of the frozen skin samples

or from TEER measurement needs to be evaporated to measure exclusively the water loss through the skin sample. With a VapoMeter (Delfin Technologies Ltd., Finland) the TEWL was determined under closed chamber conditions (Imhof et al., 2009). For this end the donor compartment of the diffusion cell was covered completely with the VapoMeter. The standard limit

of 10 g m−2 h−1 was used (Schäfer and Redelmeier, 1996b). To determine the absorption characteristics of tritiated, 3H-labeled, water, the receptor compartment was filled with physiological saline. An infinite dose (300 μl cm−2) with a specific radioactivity of 123 kBq ml−1 was applied to the surface of the skin. At distinct time points (0.5, 1, 2, 3, 4 and 5 h) receptor fluid was collected using a syringe. After the last sampling the skin was thoroughly washed with distilled water and cotton swabs. Receptor fluid was diluted with scintillation cocktail, measured by LSC and data were used to calculate the permeability constant (Kp) as described Selleck MK-2206 in Section 2.3. A generally accepted limit value of 2.5 ∗ 10−3 cm h−1 was used (Bronaugh et al., 1986). Using TWF as a pre-test, the radioactivity needs to be removed from the system before application of the test compound. Therefore, the receptor fluid was changed several times until the activity in a receptor fluid aliquot declined to 50 dpm (0.8 Bq). A 3H-labeled internal Celastrol reference standard was added to the 14C-labeled test compound formulation and applied to the skin (see Table 1 and Table 3). The concentration was determined by the specific radioactivity of the 3H-ISTD which was

chosen to be equal to the specific radioactivity of the 14C-labelled test compound (Table 1). In all samples 3H-activity was measured along with the 14C-activity by LSC. Absorption characteristics (AD and maxKp) were determined analogously, as described in Section 2.3. Following the final washing procedure at the end of the absorption experiment, 250 μl of methylene blue, 0.025% aqueous solution, was applied on top of the skin for 0.5 h and washed off with 0.7% aqueous Texapon® N70 solution. The receptor fluid was tested for permeated dye using a photometer operating at 661 nm. The concentration in the receptor fluid was determined via a calibration curve. Any staining of the epidermis was reported before digestion and processing for LSC measurements.

001, data combined over the 7 months). Soil dilution amendment did not affect plant growth and there learn more were no significant interactions between the factors (dilution, AMF, month of harvest). In the T-RFLP analysis, 68 bacterial TRFs (terminal restriction fragments) were observed

in total: Over the 7 month period 14 TRFs were present in all treatments (i.e. in bare soil, mycorrhizal and non-mycorrhizal planted soils at both dilution treatments across all harvests); 13 TRFs were present only in soils treated with the 10−1 dilution of soil slurry and absent from the 10−6 dilution treatments (planted and unplanted combined) and 14 TRFs were present in the planted treatments and absent from the macrocosms containing bare soil (dilution treatments combined). Six bacterial TRFs were associated with the planted arbuscular mycorrhizal (AM) treatment but not with the planted non-mycorrhizal (NM) treatment. A greater number of fungal TRFs were observed overall (97 TRFs): AZD6244 price over the 7 month period 15 fungal TRFs were present in all treatments; 28 TRFs were observed in planted macrocosms but not in those containing bare soil and 10 fungal TRFs were observed in the planted AM treatments compared

to the planted NM macrocosms. Of the fungal TRFs, 17 were present in soil treated with the 10−1 soil slurry dilution but absent from the 10−6 treatments. In any one dilution/planting regime per month, an overall average (grand mean) of 11 bacterial and 12 fungal TRFs were observed in sufficient

abundance to be included in the analysis. The number of bacterial TRFs identified (TRF richness) was lower in the bare unplanted and the NM planted soils amended with the 10−6 dilution than in the equivalent treatments amended with the 10−1 soil dilution one month after the experiment was established. This trend became less clear over the duration of the investigation until after 7 months the effect of dilution treatment was no longer evident, although TRF richness in the Nintedanib (BIBF 1120) NM soils was greater than in the soil which had AM fungi present (ANOVA: dilution × planting regime × month effect, F6,50 = 3.72, P = 0.004, LSD = 6.3, Fig. 2a). In months 3 and 5, the number of TRFs in the 10−1 AMF treatment was greater than in the 10−6 AMF treatment (data not shown) but by month 7 differences had disappeared ( Fig. 2a). Fungal TRF richness followed similar trends ( Fig. 2b) although data were more variable. Unplanted (bare) soil contained fewer fungal TRFs than planted soils (planting regime, F2,47 = 5.03, P = 0.010) overall. The number of fungal TRFs remained constant over all 7 months whereas the number of bacterial TRFs fell from an average (across all treatments) of 16 in month one to an average of 10 in month 7 (month as a single factor, F3,50 = 15.62, P < 0.001). PCA analysis of the microbial communities illustrated the complexity of these interactive effects.

Lumbar 5 vertebrae were scanned at a resolution of 5 μm. The X-ray tube was operated at 41 kV and 240 μA. A lower grey threshold value of 81 and upper grey threshold value of 252 was used as thresholding values in each trabecular bone sample. A cylindrical region of interest (150 slices or 0.774 mm) was selected from the centre of each vertebral body. Calibration

of the standard unit of X-ray CT density from Hounsfield units to volumetric bone mineral density (vBMD) was conducted. ROIs were analysed for the following parameters: PD98059 price trabecular thickness, trabecular separation, trabecular bone volume, trabecular porosity, as well as degree of anisotropy (DA) and structure model index (SMI). Right tibial and femoral shafts from each comparison group were subjected click here to mechanical testing (three point bending and microindentation tests respectively) after the μCT. The mechanical tests were designed to test the cortical part of bone. The tests were performed using a Zwick/Roell z2.0 testing machine (Leominster, UK) with a 100 N load cell [32]. Tibias were placed on the lower supports, at 8 mm separation, with the posterior surface of the tibia facing down. Load was applied with a loading rate of 0.1 mm s− 1 on the shaft of the tibia using the Zwick/Roell testing machine until the fracture occurred (Fig. 3A). Data were

analysed to determine values of stiffness, ultimate load and Young’s modulus using the following formula: equation(1) Young’smodulus=stiffτ⋅Ls348⋅Iwhere stiffτ is the stiffness. Ls is the separation of the supports and I is the second moment of area of the tibias. The stiffness was calculated by measuring the slope of the force-displacement graph and the ultimate load by measuring the maximum force that the bone was able to resist. The second moment of area was calculated using the microCT data and ImageJ software v1.47 and the plug-in Bone J. The micro indentation

hardness test was performed unless on equivalent transverse distal mid-shaft sections of right femur for each mouse/genotype. Bone sections were air dried and embedded in metallurgical mounting resin (EPO Set Resin, Meta Prep, UK) and the moulds allowed to solidify at room temperature for 24 h. The bone cross-section surface was subsequently polished using silicon carbide papers with decreasing grain size (240, 400, 600, 800, 1200) and diamond paste (15, 6 and 1 μm) to produce a smooth surface. After the sample preparation, micro hardness testing was performed using a Wilson Wolport Micro-Vickers 401MVA machine (UK), with an applied load of 25 g for 100 sec. The bone was tested at seven points for each specimen (Fig. 4A). The Vickers pyramid hardness number (HV) was calculated using Eq. 2 where the load (L) is in grammes force and the average length of the two diagonals (D) is in millimetres: equation(2) HV=1.854LD2. The femoral neck fracture test was used to test the mechanical properties of the femoral neck.

Under such experimental conditions, the test material

is aerosolised Obeticholic Acid mw applying high shear stresses and mass median aerodynamic diameters [MMAD] range significantly below 10 μm (ideally 1–3 μm). The respirable fraction then accounts for more than 80 vol%. In conclusion, the toxicologically relevant, respirable fraction is much lower in the products under normal handling and use conditions than under experimental conditions. Surface-treated SAS may be used in perfumes, and hence may be aerosolised during use by consumers (Becker et al., 2009). With typical aerosol particle diameters in the 10–100 μm range, most aerosol particles will not be respirable, but deposited in the nasopharyngeal region. Oral and dermal SAS exposure may arise from the use of personal care products and medicines. Recently, Dekkers et al. (2010) analysed food products

with added silica (E551), and estimated the likely oral intake of “nanosilica” via food. The authors estimated a daily intake of 124 mg “nanosilica”, corresponding to 1.8 mg/kg bw/day for an adult of 70 kg based on products containing E551, although it is stated in the publication C59 mouse itself that “… it is not clear whether the food additive E551 contains nano-sized silica.” The terminology “nanosilica” as used by Dekkers et al. (2010) was later criticized by Bosch et al. (2011). Silica is usually tightly bound into the matrix of end-use articles, and hence significant exposure of the general population through these products is unlikely. The different forms of SAS have been used as test materials

in a number of environmental, ecotoxicological and toxicological studies. Some of these studies were conducted to investigate the toxic potential of SAS while others used SAS as a comparison material in studies on various nanoparticles. Several studies described in the following sections refer to the testing of “nanosilica” versus “bulk silica”, with some studies highlighting the enhanced biological responses for nano-forms versus the findings for larger silica particles. selleck screening library These studies, however, generally refer to the primary particle diameter when classifying some silica products as “nano” rather than a whole-particle dimension that reflects the complex aggregate structures of most silica particles, such as the aggregate diameter. This can lead to the misinterpretation of these study findings as reflecting an effect of particle size while it is well known that silica particles can differ in other toxicologically relevant properties, such as surface area and particle number. Pyrogenic, precipitated and gel forms of SAS, including surface-treated forms, have been the subject of dissolution testing using a simulated biological medium at 37 °C and pH values near 7 (Roelofs and Vogelsberger, 2004). Depending on the material, the solubility was between 2.3 and 2.

Both samples were loaded in a Phenomenex C18 column (Jupiter 5 μ, 4 × 150 mm, California, USA) in a two-solvent system: (A)

trifluoroacetic acid (TFA)/H2O (1:1000) and (B) TFA/Acetonitrile (ACN)/H2O (1:900:100). The column was eluted at a flow rate of 1 mL/min with a 10–80% gradient of solvent B over 40 min. The HPLC column eluates were monitored by their absorbance at 214 nm. The peptides eluted were analyzed on a MALDI-ToF/PRO instrument (G&E Healthcare – Sweden). Samples were mixed 1:1 (v: v) with a supersaturated solution matrix for peptides (α-cyano 4-hydroxycinnamic acid in 50% acetonitrile containing 0.1% TFA), deposited on the sampling plate (0.4–0.8 l) and dried. The spectrometer was operated in reflectron mode and P14R ([M + H+] + 1533.85) and angiotensin II ([M + H+] + 1046.54)

(Sigma, St. Louis, MO) were used as external calibrants. SDS-PAGE was carried out according to the method of Laemmli (1970). Sting Crenolanib purchase venom, skin mucus and protein fractions (10 μg) of C. spixii were analyzed by SDS-PAGE Panobinostat 4–20% acrylamide gradient under reducing conditions. Prior to electrophoresis, the samples were mixed 1:1 (v/v) with sample buffer. The gel was stained with the Silver method. For protein deglycosylation under denaturing conditions, toxin samples (20 μg) were incubated in 10% SDS for 1 min at 95 °C. After adding 0.02 M sodium phosphate buffer, 0.08% sodium azide, 0.01 M EDTA, 2% Triton X-100, pH 7.0, incubation was prolonged for 2 min at 95 °C. After cooling, 1 U of N-glycosidase F (Roche, Mannheim, Germany) was added, and the mixture was incubated for 1 h at 37 °C. The deglycosylation profiles were evaluated by SDS-PAGE as described above. The protein Fv6 was reduced and alkylated with 4-vinyl pyridine as described (Wilson and Yuan,

1989). One milligram-aliquots of Fv6 were dissolved in 1 ml of 0.1 M Tris–HCl (pH 8.6), 6 M guanidine-HCl. After addition of 30 μL β-mercaptoethanol the samples were incubated first at 50 °C for 4 h under nitrogen, then after addition of 40 μL of 4-vinyl pyridine, in the dark at 37 °C for 2 h and subsequently desalted on a PD-minitrap G25 column. The S-pyridylethylated proteins were cleaved with 2% (w/w) chymotrypsin at 37 °C for 3 h. The cleavage products were separated on a Vydac C18 small pore column (4.6 × 250 mm) Y-27632 2HCl in a linear gradient of 0–50% acetonitrile in 0.1% aqueous TFA and sequenced using a Shimadzu PPSQ-21A protein sequencer. The partial primary structure of Fv6 was compared with the sequences of other related proteins in the SWISS-PROT/TREMBL data bases using the FASTA 3 and BLAST programs. The dynamics of alterations in the microcirculatory network were determined using intravital microscopy by transillumination of mice cremaster muscle after subcutaneous application of 10 μg of all fractions, sting venom or skin mucus of C. spixii dissolved in 20 μL of sterile saline. Administration of the same amount of sterile saline was used as control.

24 Digital images were analysed using Sigma-Scan 2.0 software. The distance between the cemento–enamel junctions up to the height of alveolar bone of the first mandibular molar on the mesial side of the rat was recorded. Samples were homogenised in Trizol reagent (Invitrogen) for 1 min using a tissue homogenizer (Polytron-Agrgregate, Kinematica, Littau/Luzern, Switzerland) at maximum speed. Total RNA was isolated according to the manufacturer’s guidelines and quantified by a spectrophotometer. KU-57788 molecular weight The integrity of RNA was verified by agarose gel electrophoresis. Complementary DNA was prepared using 2 μg of total RNA and a reverse transcriptase. The primers used

in the experiments were the standard TaqMan (Applied Biosystems, Foster City, CA, USA) brand. The gene analysed were TNF-α

(primers: sense 5′ GGC ATG GAT CTC AAA GAC AAC C-3′ and antisense 5′-CAA ATC GGC TGA CGG TGT G-3′). Glyceraldehyde-3-phosphate dehydrogenase (GenBank NM_017008) was used as a housekeeping gene. Real-time polymerase chain reaction was carried out in the StepOne polymerase chain reaction cycler (Applied Biosystems, Foster City, CA, USA). The polymerase chain reaction conditions were 95 °C for 10 min, followed by 40 cycles at 95° for 10 s and 60 °C for 45 s. Real-time data were analysed using the Sequence Detector System 1.7 (Applied Biosystems, Foster City, CA, USA). Results are expressed as fold inductions compared with controls. Results GSK J4 nmr are presented as means ± SEM for the number of rats (n) indicated. The data were analysed by the unpaired Student’s t-test for two mean comparisons and one-way ANOVA (with Bonferroni post hoc test) for bone reabsorption and TNF-α expression. The level of significance was set at P < 0.05. Table 1 shows that the body weight and naso-anal length were 29% and 15% respectively, RANTES lower in MSG groups when compared with CTL (P < 0.05), however, the Lee Index was 8% higher in the MSG rats (P < 0.003). The retroperitoneal and perigonadal fat pads weight doubled in

MSG rats when compared with CTL rats (P < 0.0001, Fig. 1A and B). The neonatal MSG treatment did not influence the plasma concentration of glucose, NEFA and total CHOL (P > 0.05). However, in the MSG group plasma and TG concentrations were 3.0 and 4.0 times higher (P < 0.0001 and P < 0.0002), respectively than, CTL group ( Table 2). According to Fig. 2, alveolar bone resorption was 44% lower in obese-MSG group compared with CTL group (P < 0.01). In the presence of ligature, there was a significant increase in alveolar bone resorption in both groups CTL L and MSG L compared with CTL and MSG group respectively (P < 0.001). However, alveolar bone resorption in the MSG L animals was similar to that occurring in the CTL group (P > 0.05) ( Fig. 3A–D). The TNF-α gene expression in periodontal tissue was similar in MSG and CTL animals in the absence of ligature (P > 0.

After cooling, the extracts were centrifuged at 8000 × g for 20 min. The collected supernatants were filtered with qualitative filter papers (Whatman) and transferred to glass flasks at 40 °C until solvent was completely evaporated (approximately 72 h). The dry glucosinolate-containing precipitate was reconstituted with 1 mL of 0.2 mol L−1 HEPES–KOH AZD2281 cell line (pH 7.0) in the same container. An extract aliquot (10 μL), which was previously reconstituted in 0.2 mol L−1 HEPES–KOH (pH 7.0), was incubated with 5 μL of a thioglucosidase solution

(0.12 U). The thioglucosidase solution contained myrosinase purified from Sinapis alba L. (Sigma–Aldrich), which was buffered in 0.2 mol L−1 HEPES–KOH (pH 7.0) at 37 °C for 24 h; this procedure was in accordance with the methodology of Li and Kushad (2005) which was performed in 3 mL test tubes. In agreement with the degradation reaction of glucosinolates by thioglucosidase, the measurement is accomplished on glucose produced upon glucosinolate hydrolysis. Glucosinolate content was quantified according to the stoichiometry proposed by Palmieri, Iori, and Leoni (1987), which states that 1 mol of released glucose is

equivalent to 1 mol of Tenofovir clinical trial total glucosinolate. The enzymatic catalysis was stopped with the addition of 5 μL of 18 mmol L−1 perchloric acid solution (HClO4). To detect the background levels of glucose in the samples, a control was prepared. The control contained buffered extract (10 μL) with 18 mmol L−1 HClO4 (5 μL), and 5 μL of the thioglucosidase solution was rapidly added. The liberated total glucose was assayed enzymatically by using a glucose oxidase/peroxidase kit (CELM, Brazil). Sinigrin, an allyl-glucosinolate (Sigma), was used

as a calibrant and as a positive control. The sample extraction procedure was identical to the one described for total glucosinolates (n = 3, each in triplicate). The extracts were filtered on Millex™ polyvinylidene fluoride (PVDF) membranes (0.45 μm, Millipore) prior to HPLC injection. The methodology used for the determination of benzylglucosinolate was described by Kiddle et al. (2001) and modified by Rossetto et al. (2008). The calibration curve for benzylglucosinolate and the internal standardization for the sample recovery test were carried out according to Rossetto et al. (2008). A single chromatographic Amino acid run with an internal standard (50 μL of 12 nmol L−1sinigrinin 1 mL of 70:30 MeOH (mL):water (mL) that also contained 1.49 g L−1 TFA) was also completed to determine the sinigrin (allyl-glucosinolate) retention time. Benzylglucosinolate was isolated by HPLC, which was coupled to an automatic injector and a quaternary pump (HP 1100). The substance was detected by a diode array (PDA) detector at a spectral range of 200–400 nm. A reverse phase column (Luna C18, 250 × 4.6 mm, 5 μm) developed by Phenomenex was used, and the column was coupled to a Security Guard pre-column (Phenomenex). The column temperature was maintained at 25 °C.