A transcriptomic signature to predict adjuvant gemcitabine sensitivity in pancreatic adenocarcinoma.

BACKGROUND: Chemotherapy is the only systemic treatment approved for pancreatic ductal adenocarcinoma (PDAC), with a selection of regimens based on patients' performance status and expected efficacy. The establishment of a potent stratification associated with chemotherapeutic efficacy could potentially improve prognosis by tailoring treatments. PATIENTS AND METHODS: Concomitant chemosensitivity and genome-wide RNA profiles were carried out on preclinical models (primary cell cultures and patient-derived xenografts) derived from patients with PDAC included in the PaCaOmics program (NCT01692873). The RNA-based stratification was tested in a monocentric cohort and validated in a multicentric cohort, both retrospectively collected from resected PDAC samples (67 and 368 patients, respectively). Forty-three (65%) and 203 (55%) patients received adjuvant gemcitabine in the monocentric and the multicentric cohorts, respectively. The relationships between predicted gemcitabine sensitivity and patients' overall survival (OS) and disease-free survival were investigated. RESULTS: The GemPred RNA signature was derived from preclinical models, defining gemcitabine sensitive PDAC as GemPred+. Among the patients who received gemcitabine in the test and validation cohorts, the GemPred+ patients had a higher OS than GemPred- (P = 0.046 and P = 0.00216). In both cohorts, the GemPred stratification was not associated with OS among patients who did not receive gemcitabine. Among gemcitabine-treated patients, GemPred+ patients had significantly higher OS than the GemPred-: 91.3 months [95% confidence interval (CI): 61.2-not reached] versus 33 months (95% CI: 24-35.2); hazard ratio 0.403 (95% CI: 0.221-0.735, P = 0.00216). The interaction test for gemcitabine and GemPred+ stratification was significant (P = 0.0245). Multivariate analysis in the gemcitabine-treated population retained an independent predictive value. CONCLUSION: The RNA-based GemPred stratification predicts the benefit of adjuvant gemcitabine in PDAC patients.

Patient outcome according to the 2017 international consensus on the definition of borderline resectable pancreatic ductal adenocarcinoma.

BACKGROUND/OBJECTIVE: We evaluated the usefulness of the 2017 definition of borderline pancreatic ductal adenocarcinoma (BR-PDAC) in fit patients (performance status 0-1) based on anatomical (A) and biological dimensions (B). METHODS: From 2011 to 2018, 139 resected patients with BR-PDAC according to the 2017 definition were included: 18 patients underwent upfront pancreatectomy (CA 19-9 > 500 U/mL and/or regional lymph node metastasis; BR-B group), and 121 received FOLFIRINOX (FX) induction chemotherapy and were divided into BR-A (CA 19-9 < 500 U/mL, no regional lymph node metastasis; n = 68) and BR-AB (CA 19-9 > 500 U/mL and/or regional lymph node metastasis; n = 53) groups. RESULTS: The 3 groups were comparable according to patient characteristics (except for back pain (P < .01) and CA 19-9 (P < .01)), intraoperative data, and postoperative courses. BR-AB patients required more venous resections (P < .01). The 3 groups were comparable on pathologic findings, except that BR-B patients had more lymph node invasions (P = .02). Median overall survival (OS) of the 121 patients was 45 months. In multivariate analysis, venous resection (P = .039) and R1 resection (P = .012) were poorly linked with OS, whereas BR-A classification (P < .01) independently favored OS. Median survival times of BR-A, BR-AB, and BR-B groups were undetermined, 27 months, and 20 months (P < .001), respectively. CONCLUSIONS: The 2017 definition was relevant for sub-classifying patients with BR-PDAC. The anatomical dimension (BR-A) was a favorable prognostic factor, whereas the biological dimension (BR-AB and BR-B) poorly impacted survival.

Has the non-resection rate decreased during the last two decades among patients undergoing surgical exploration for pancreatic adenocarcinoma?

PURPOSE: To determine if improvement in imaging reduces the non-resection rate (NRR) among patients with pancreatic ductal adenocarcinoma (PDAC). METHODS: From 2000 to 2019, 751 consecutive patients with PDAC were considered eligible for a intention-to-treat pancreatectomy and entered the operating room. In April 2011, our institution acquired a dual energy spectral computed tomography (CT) scanner and liver diffusion weighted magnetic resonance imaging (DW-MRI) was included in the imaging workup. We consequently considered 2 periods of inclusion: period #1 (February 2000-March 2011) and period #2 (April 2011-August 2019). RESULTS: All patients underwent a preoperative CT scan with a median delay to surgery of 18 days. Liver DW-MRI was performed among 407 patients (54%). Median delay between CT and surgery decreased (21 days to 16 days, P < .01), and liver DW-MRI was significantly most prescribed during period #2 (14% vs 75%, P < .01). According to the intraoperative findings, the overall NRR was 24.5%, and remained stable over the two periods (25% vs 24%, respectively). While vascular invasion, liver metastasis, and carcinomatosis rates remained stable, para-aortic lymph nodes invasion rate (0.4% vs 4.6%; P < 0.001) significantly increased over the 2 periods. The mean size of the bigger extra pancreatic tumor significantly decrease (7.9 mm vs 6.4 mm (P < .01), respectively) when the resection was not done. In multivariate analysis, CA 19-9 < 500 U/mL (P < .01), and liver DW-MRI prescription (P < .01) favoured the resection. CONCLUSIONS: Due to changes in our therapeutic strategies, the NRR did not decrease during two decades despite imaging improvement.

Buccodental side effects of sunitinib in patients with metastatic renal cell carcinoma.

BACKGROUND: Sunitinib is a tyrosine kinase inhibitor approved for the treatment of renal cell carcinoma (RCC). Few data evaluated severe buccodental adverse events. The aim of this study was to evaluate sunitinib buccodental toxicity in patients with metastatic RCC and to compare it with that of standard chemotherapy in patients with other solid cancers. METHODS: Patients with RCC treated with sunitinib and patients with other solid tumours treated with chemotherapy were followed for 3 months. Data on dental appliances, oral hygiene/care practices before and during treatment were collected. RESULTS: A total of 116 patients were included (58 RCC treated by sunitinib: group S, and 58 treated by chemotherapy: group C). No differences in dental care habits were noted before treatment. In group S, patients reported significantly more frequent pain (P<0.01), teeth instability (P=0.01), gingival bleeding (P=0.01) and change in teeth colour (P=0.02). In all, 58% of patients in this group had to modify their diet (P<0.01). Frequency of dentist visits for teeth removal was increased (25% vs 8%, P=0.01). CONCLUSION: Sunitinib seems to increase buccodental toxicity as compared with chemotherapy. This finding emphasises the need for optimal dental care and standardised dental follow-up in patients treated with sunitinib.

Drug-drug interactions with proton pump inhibitors in cancer patients: an underrecognized cause of treatment failure.

New concepts and drugs have revolutionized medical treatment for cancers. These drugs, which are very expensive and usually well tolerated, have dramatically improved cancer prognosis. We must use them wisely for patients to fully benefit. Gastric acid antisecretory drugs and particularly proton pump inhibitors (PPIs) revolutionized the treatment of gastroduodenal ulcers and severe gastroesophageal reflux, but are frequently overused for symptomatic treatment of epigastric pain or heartburn. Long-term acid suppression may alter the efficacy of many anticancer drugs, such as tyrosine kinase inhibitors (TKIs), cyclin-dependent kinase (CDK) 4/6 inhibitors and immune checkpoint inhibitors (ICIs), by either decreasing gastric acid secretion and thus drug absorption, or by modifying the gut microbiome that modulates the response to ICIs. Oncologists thus need to pay particular attention to the concomitant use of PPIs and anticancer drugs. These interactions translate into major clinical impacts, with demonstrated loss of efficacy for some TKIs (erlotinib, gefitinib, pazopanib), and conflicting results with many other oral drugs, including capecitabine and CDK 4/6 inhibitors. Furthermore, the profound changes in the gut microbiome due to using PPIs have shown that the benefit of using ICIs may be suppressed in patients treated with PPIs. As the use of PPIs is not essential, we must apply the precautionary principle. The first sentence of a recent Comment in Nature was "Every day, millions of people are taking medications that will not help them". We fear that every day millions of cancer patients are taking medications that harm them. While this may well be only association and not causation, there is enough to make us pause until we reach a clear answer. All these data should encourage medical oncologists to refrain from prescribing PPIs, explaining to patients the risks of interaction in order to prevent inappropriate prescription by another physician.

BAYPAN study: a double-blind phase III randomized trial comparing gemcitabine plus sorafenib and gemcitabine plus placebo in patients with advanced pancreatic cancer.

BACKGROUND: Sorafenib is an oral anticancer agent targeting Ras-dependent signaling and angiogenic pathways. A phase I trial demonstrated that the combination of gemcitabine and sorafenib was well tolerated and had activity in advanced pancreatic cancer (APC) patients. The BAYPAN study was a multicentric, placebo-controlled, double-blind, randomized phase III trial comparing gemcitabine/sorafenib and gemcitabine/placebo in the treatment of APC. PATIENTS AND METHODS: The patient eligibility criteria were locally advanced or metastatic pancreatic adenocarcinoma, no prior therapy for advanced disease and a performance status of zero to two. The primary end point was progression-free survival (PFS). The patients received gemcitabine 1000 mg/m(2) i.v., weekly seven times followed by 1 rest week, then weekly three times every 4 weeks plus sorafenib 200 mg or placebo, two tablets p.o., twice daily continuously. RESULTS: Between December 2006 and September 2009, 104 patients were enrolled on the study (52 pts in each arm) and 102 patients were treated. The median and the 6-month PFS were 5.7 months and 48% for gemcitabine/placebo and 3.8 months and 33% for gemcitabine/sorafenib (P = 0.902, stratified log-rank test), respectively. The median overall survivals were 9.2 and 8 months, respectively (P = 0.231, log-rank test). The overall response rates were similar (19 and 23%, respectively). CONCLUSION: The addition of sorafenib to gemcitabine does not improve PFS in APC patients.

A continuous spectrophotometric assay for determination of the aureusidin synthase activity of tyrosinase.

INTRODUCTION: Aurones (aureusidin glycosides) are plant flavonoids that provide yellow colour to the flowers of some ornamental plants. In this study we analyse the capacity of tyrosinase to catalyse the synthesis of aureusidin by tyrosinase from the chalcone THC (2',4',6',4-tetrahydroxychalcone). OBJECTIVE: To develop a simple continuous spectrophotometric assay for the analysis of the spectrophotometric and kinetic characteristics of THC oxidation by tyrosinase. METHODOLOGY: THC oxidation was routinely assayed by measuring the increase in absorbance at 415 nm vs. reaction time. RESULTS: According to the mechanism proposed for tyrosinase, the enzymatic reaction involves the o-hydroxylation of the monophenol THC to the o-diphenol (PHC, 2',4',6',3,4 - pentahydroxychalcone), which is then oxidised to the corresponding o-quinone in a second enzymatic step. This product is highly unstable and thus undergoes a series of fast chemical reactions to produce aureusidin. In these experimental conditions, the optimum pH for THC oxidation is 4.5. The progress curves obtained for THC oxidation showed the appearance of a lag period. The following kinetic parameters were also determined: K(m )= 0.12 mM, V(m )= 13 microM/min, V(m)/K(m )= 0.11/min. CONCLUSION: This method has made it possible to analyse the spectrophotometric and kinetic characteristics of THC by tyrosinase. This procedure has the advantages of a short analysis time, straightforward measurement techniques and reproducibility. In addition, it also allows the study of tyrosinase inhibitors, such as tropolone.

Partial purification, characterisation and histochemical localisation of alkaline phosphatase from ascocarps of the edible desert truffle Terfezia claveryi Chatin.

In the present paper, we confirmed that alkaline phosphatase (ALP) is the main phosphatase present in ascocarps of the edible mycorrhizal fungus Terfezia claveryi. The enzyme was partially purified by precipitation with polyethylene glycol. The purification achieved from a crude extract was fivefold, with 53% of the activity recovered, and acid phosphatase, most of the lipids and phenolic compounds were eliminated. Alkaline phosphatase was kinetically characterised at pH 10.0, the optimum for this enzyme, using p-nitrophenyl phosphate as substrate. The V(max) and K(m) values were 0.3 micromol.min(-1).mg(-1) protein and 9.0 mM, respectively. Orthovanadate was a competitive inhibitor of ALP, with a K(i) of 42.5 microM. The enzyme was histochemically localised in the peridium, the hypothecium and in the ascogenic hyphae of the gleba using both colour and fluorescent reactions. The results presented suggest that the ascocarp of T. claveryi, at some stages of its development, may become nutritionally autonomous and independent of the host plant.

Outcomes of pancreatic adenocarcinoma that was not resected because of isolated para-aortic lymph node involvement.

PURPOSE: Survival appears to be poor in cases of pancreatic ductal adenocarcinoma (PDAC) with para-aortic lymph node involvement (PALN+). However, resection is still performed in these cases because the prognostic impact of PALN+remains controversial. METHODS: PALN+was intraoperatively found in 14 patients (4.8%) with resectable PDAC who consequently did not undergo pancreatectomy. RESULTS: The median overall survival time after laparotomy was 21 months. The 1- and 3-year overall survival rates were 58.3% and 25%, respectively. CONCLUSIONS: We support the advisability of reconsidering pancreatectomy in patients with intraoperatively detected PALN+because the reported survival of such patients who undergo pancreatectomy is poorer than the survival observed for patients in our series.

Resectable pancreatic head adenocarcinoma: Is R0 resection an illusion? Genetic evaluation of venous resection margin affirmed unrecognized disease.

PURPOSE: To assess the K-ras gene mutation in the histologically negative venous margin of a pancreaticoduodenectomy (PD) specimen and its impact on survival. METHOD: From 2007 to 2010, 22 patients underwent R0 PD for resecable pancreatic adenocarcinoma. All specimens were stained and the portal vein (PV) bed was identified by blue ink; a 2mm3 sample (including the blue ink) was cut from a microscopic free-tumor block. DNA was extracted and assessed by quantitative real time polymerase chain reaction to detect the K-ras gene mutation. Twelve specimens (55%) (kras+ group) were identified with a K-ras mutation in the venous margin resection, and 10 specimens (kras- group) did not have K-ras mutation detected in the venous margin resection. RESULTS: The two groups were comparable. Overall 3years survival of patients of kras+ group versus patients of kras- group was 0 and 17% (P=0.03), respectively. Median survival time of patients of kras+ group versus patients of kras- group was 16months vs 25months (P=0.04; 95% confidence interval [1,11-1,88]), respectively. CONCLUSION: Genetic evaluation of venous resection margin affirmed unrecognized disease with strong impact on survival in more than 50% of patients with histologically R0 resection.

Hydroperoxidase activity of lipoxygenase: a kinetic study of isoproterenol oxidation.

The hydroperoxidase activity of soybean lipoxygenase, a non-heme protein, oxidized isoproterenol using H2O2 at pH 6.0. This oxidation was enzymatic, since neither heat-denaturated enzyme or iron ions in the presence of H2O2 produced an increase in absorbance. The initial rate was not linear and showed a characteristic lag period whose length depended on the enzyme and substrate concentration. The lag was decreased if the enzyme and isoproterenol concentration were increased, whereas it increased if the H2O2 concentration was increased. Lipoxygenase showed the typical low specificity for electron donor characteristic of this hydroperoxidase activity (26 mM), but a high affinity for H2O2 (94 microM), although with substrate inhibition (ksi = 3.6 mM). The chemical intermediates produced during the oxidation of isoproterenol were characterized in order to determine the origin of the lag period. A plausible kinetic mechanism is proposed to explain the observed lag period and inhibition by high concentrations of H2O2.

Lipoxygenase-catalyzed oxidation of chlorpromazine by hydrogen peroxide at acidic pH.

The hydroperoxidase activity of soybean lipoxygenase, a non-heme protein, oxidizes chlorpromazine using H2O2 at acidic pHs ranging from 3.0 to 4.0. The enzyme is assayed at pH 3.5, at which the half-life is 2 h (lower pHs cause higher inactivation rates). This oxidation is enzymatical since boiled enzyme or even iron ions both with H2O2 failed to produce any increase in absorbance. In addition, the concentration of CPZ radical cation formed and the concomitant enzyme activity directly depends on the enzyme concentration up to 0.23 microM. The Vmax value is 125 mumol/min per mg protein and the Km for chlorpromazine and H2O2 are 2.1 mM and 0.25 mM, respectively. Similar results were obtained when linoleic acid hydroperoxide was used instead of H2O2 with a Km value of 95 microM. The radical cation obtained in the oxidation of chlorpromazine by lipoxygenase decays by a disproportionation reaction. This permits to consider the overall reaction as a sum of an enzymatic reaction coupled with a chemical second order reaction with substrate regeneration, similar to those produced by peroxidases from different sources.

Sesqui- and triterpenoids from the liverwort Lepidozia chordulifera inhibitors of bacterial biofilm and elastase activity of human pathogenic bacteria.

Five dammarane-type triterpenoids, five pentacyclic triterpenoids (three of them carrying a carboxylic acid group), and two aromadendrane-type sesquiterpenoids were isolated from an Argentinian collection of the liverwort Lepidozia chordulifera. Compounds were characterized by comparison of their spectral data with those previously reported and tested in their ability to control bacterial growth, biofilm formation, bacterial Quorum Sensing process (QS), and elastase activity of Pseudomonas aeruginosa, as well as bacterial growth and biofilm formation of Staphylococcus aureus. The key role played by biofilm and elastase activity in bacterial virulence make them a potential target for the development of antibacterial agents. The aromadendrane-type sesquiterpenoid viridiflorol was the most potent biofilm formation inhibitor, producing 60% inhibition in P. aeruginosa and 40% in S. aureus at 50µg/ml. Ursolic and betulinic acids (two of the pentacyclic triterpenoids isolated) were able to reduce 96 and 92% the elastase activity of P. aeruginosa at 50µg/ml, respectively. Among the analyzed triterpenoids, those that carry a dammarane skeleton were the most potent inhibitors of the P. aeruginosa biofilm formation and were active against both P. aeruginosa and S. aureus. Subsequently, a computer-assisted study of the triterpenoid compounds was carried out for a better understanding of the structure-activity relationships.

Oxidation of aminopyrine by the hydroperoxidase activity of lipoxygenase: a new proposed mechanism of N-demethylation.

The oxidation of aminopyrine, an N-alkyl aromatic amine, by the hydroperoxidase activity of lipoxygenase was studied. Aminopyrine gave rise to a purple color in the presence of H2O2 and lipoxygenase, the color being proportional to the aminopyrine radical cation. The H2O2/aminopyrine radical cation molar ratio was 0.5. The overall reaction was considered as an enzymic-chemical second order mechanism with substrate regeneration. From the equations, the apparent constant of the radical cation's decomposition (k'app) was evaluated under different experimental conditions. It was found to be inversely proportional to the proton concentration but unaffected by the concentration of aminopyrine. These results suggest a new comprehensive mechanism for N-demethylation, which takes into account the described presence of both nitrogen- and carbon-centered radicals and the marked effect of pH on the stability of the radical cation.

Enzymatic oxidation of phenothiazines by lipoxygenase/H2O2 system.

Lipoxygenase (LOX) (EC 1.13.11.12) oxidized a wide range of phenothiazine (Pt) tranquillizers to their corresponding radical cations in the presence of H2O2 by means of an enzymatic chemical second-order mechanism with substrate regeneration similar to that of horseradish peroxidase. The optimum pH of LOX for this hydroperoxidase activity was in the acid range (pH 3.0-4.0), as has been shown for other Pt oxidizing systems, such as peroxidase/H2O2 and haemoglobin. LOX showed Michaelis constants for Pt ranging from 1.4 to 8.5 mM and which, in some cases, e.g. trifluoperazine, displayed substrate inhibition. By contrast, it had a high affinity for H2O2 in the microM to mM range. A new, previously undescribed plot, which relates the enzymatic affinity and the apparent second-order decay of the cation radical, was developed to study the influence of the 2- and 10-substituents in the Pt ring. The implications of this new plot and the LOX-mediated Pt oxidation are also discussed.

Purification, stabilization and characterization of tomato fatty acid hydroperoxide lyase.

Fatty acid hydroperoxide lyase (HPO-lyase) was purified 300-fold from tomatoes. The enzymatic activity appeared to be very unstable, but addition of Triton X100 and beta-mercaptoethanol to the buffer yielded an active enzyme that could be stored for several months at -80 degrees C. The enzyme was inhibited by desferoxamine mesylate (desferal), 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone), nordihydroguaiaretic acid (NDGA), n-propyl gallate and butylated hydroxyanisole, suggesting the involvement of free radicals in the reaction mechanism and the existence of a prosthetic group in the active center. However, no heme group could be demonstrated with the methods commonly used to identify heme groups in proteins. Only 13-hydroperoxides from linoleic acid (13-HPOD) and alpha-linolenic acid (alpha-13-HPOT) were cleaved by the tomato enzyme, with a clear preference for the latter substrate. The pH-optimum was 6.5, and for concentrations lower than 300 microM a typical Michaelis-Menten curve was found with a K(m) of 77 microM. At higher alpha-13-HPOT concentrations inhibition of the enzyme was observed, which could (at least in part) be attributed to 2E-hexenal. A curve of the substrate conversion as a function of the enzyme concentration revealed that 1 nkat of enzyme activity converts 0.7 mumol alpha-13-HPOT before inactivation. Headspace analysis showed that tomato HPO-lyase formed hexanal from 13-HPOD and 3Z-hexenal from alpha-13-HPOT. A trace of the latter compound was isomerized to 2E-hexenal. In addition to the aldehydes, 12-oxo-9Z-dodecenoic acid was found by GC/MS analysis. To a small extent, isomerization to 12-oxo-10E-dodecenoic acid occurred.

Rapid reversed-phase high-performance liquid chromatographic determination of the regiospecificity of lipoxygenase products on linoleic acid.

A new reversed-phase high-performance liquid chromatography method for the separation of regioisomeric products from lipoxygenase acting on linoleic acid was studied. The addition of salts to the mobile phase improved the retention and separation behaviour of 13-hydroperoxy-9,11-octadecadienoic acid and 9-hydroperoxy-10,12-octadecadienoic acid with respect to the results obtained with other mobile phases reported in the literature. The effect of the pH and ionic strength of the buffer on the retention times, capacity factor and separation factor of these lipoxygenase products were also studied. The pH optimum coincided with the pKa of linoleic acid (close to 7 depending on the fatty acid concentration). Phosphate concentrations close to 100 mM considerably reduced the retention times and led to better separation of the mixture of both products. Finally, this method was applied to the identification and separation of two linoleic acid hydroxides (13-hydroxy-9,11-octadecadienoic acid and 9-hydroxy-10,12-octadecadienoic acid) obtained by the reduction of their corresponding hydroperoxides.

Characterization of patatin esterase activity in AOT-isooctane reverse micelles.

Patatin is a family of glycoproteins that accounts for 30-40% of the total soluble protein in potato (Solanum tuberosum L.) tubers. This protein has been reported not only to serve as a storage protein but also to exhibit lipid acyl hydrolase (LAH) activity. In this study patatin is characterized in AOT-isooctane reverse micelles. The influence on the enzymatic activity of characteristic parameters of reverse micelles, w(o) (= H(2)O/AOT), and the percentage of H(2)O, theta, were investigated. The results obtained show that patatin esterase activity varies with w(o) but remains constant throughout the range of theta values studied. The variation with w(o) showed that the activity follows an S-shaped behavior pattern, reaching a maximum at about w(o) = 20 for 2% H(2)O. Patatin esterase activity was compared with p-nitrophenyl (PNP) fatty acid esters of different chain lengths. The activity was much higher for PNP-caprylate. The pH optimum was 6.0, different from the value obtained when patatin esterase activity was measured in mixed micelle systems. The optimal temperature was 35 degrees C, above which the activity decreased to almost zero. The kinetic parameters were also evaluated (K(m) = 10 mM, V(m) = 158 microM/min, V(m)/K(m) = 15.8 x 10(-3) min(-1)). This paper shows the suitability of reverse micelles for measuring patatin esterase activity, since it allows the study of the enzyme in similar conditions to that prevailing in vivo.

Characterization of catecholase and cresolase activities of eggplant polyphenol oxidase.

In the present paper the catecholase and cresolase activities of eggplant polyphenol oxidase (PPO) are described. To preserve the latter activity, a partially purified enzyme was used. Peroxidase was removed from the preparation to avoid its interference with PPO during phenol oxidation. The partially purified eggplant PPO was fully active. The catecholase/cresolase ratio of 41.1 indicated that, in a pH close to the physiological, diphenol oxidation predominates over monophenol oxidation. The characteristic lag phase of the cresolase activity is modulated by the pH, the monophenol and diphenol concentrations, and the enzyme's concentration. The effect of several inhibitors was also tested, and the K(i) values of the two most effective (tropolone and 4-hexylresorcinol) were determined.

Partial purification, characterization, and histochemical localization of fully latent desert truffle (Terfezia claveryi Chatin) polyphenol oxidase.

In the present paper, a fully latent polyphenol oxidase (PPO) from desert truffle (Terfezia claveryi Chatin) ascocarps is described for the first time. The enzyme was partially purified by using phase partitioning in Triton X-114 (TX-114). The achieved purification was 2-fold from a crude extract, with a 66% recovery of activity. The interfering lipids were reduced to 13% of the original content. In addition, the purification gave rise to a reduction of phenolic compounds to only 37.5%, thus avoiding the postpurification tanning of the enzyme. Latent PPO was activated by the anionic surfactant sodium dodecyl sulfate (SDS) or by incubation with trypsin. The amount of SDS necessary to obtain a maximum activation was dependent on the nature of the substrate. The use of SDS also permitted the histochemical localization of the latent enzyme within the ascocarp. Terfezia polyphenol oxidase was kinetically characterized using two phenolic substrates (L-DOPA and tert-butylcatechol). The latter substrate presented inhibition at high substrate concentration with a K(si) of 6.3 mM. Different inhibiting agents (kojic and cinnamic acid, mimosine and tropolone) were also studied, tropolone being the most effective.