The safener isoxadifen does not increase herbicide resistance evolution in recurrent selection with fenoxaprop.

Safeners are chemical compounds used to improve selectivity and safety of herbicides in crops by activating genes that enhance herbicide metabolic detoxification. The genes activated by safeners in crops are similar to the genes causing herbicide resistance through increased metabolism in weeds. This work investigated the effect of the safener isoxadifen-ethyl (IS) in combination with fenoxaprop-p-ethyl (FE) on the evolution of herbicide resistance in Echinochloa crus-galli under recurrent selection. Reduced susceptibility was observed in the progeny after recurrent selection with both FE alone and with FE + IS for two generations (G2) compared to the parental population (G0). The resistance index found in G2 after FE + IS selection was similar as when FE was used alone, demonstrating that the safener did not increase the rate or magnitude of herbicide resistance evolution. G2 progeny selected with FE alone and the combination of FE + IS had increased survival to herbicides from other mechanisms of action relative to the parental G0 population. One biotype of G2 progeny had increased constitutive expression of glutathione-S-transferase (GST1) after recurrent selection with FE + IS. G2 progeny had increased expression of two P450 genes (CYP71AK2 and CYP72A122) following treatment with FE, while G2 progeny had increased expression of five P450 genes (CYP71AK2, CYP72A258, CYP81A12, CYP81A14 and CYP81A21) after treatment with FE + IS. Repeated selection with low doses of FE with or without the safener IS decreased E. crus-galli control and showed potential for cross-resistance evolution. Addition of safener did not further decrease herbicide sensitivity in second generation progeny; however, the recurrent use of safener in combination with FE resulted in safener-induced increased expression of several CYP genes. This is the first report using safener as an additional factor to study herbicide resistance evolution in weeds under experimental recurrent selection.

New Oxazolidines Inhibit the Secretion of IFN-γ and IL-17 by PBMCS from Moderate to Severe Asthmatic Patients.

BACKGROUND: Moderate to severe asthma could be induced by diverse proinflammatory cytokines, as IL-17 and IFN-γ, which are also related to treatment resistance and airway hyperresponsiveness. Oxazolidines emerged as a novel approach for asthma treatment, since some chemical peculiarities were suggested by previous studies. OBJECTIVE: The present study aimed to evaluate the IL-17A and IFN-γ modulatory effect of two new oxazolidine derivatives (LPSF/NB-12 and -13) on mononucleated cells of patients with moderate and severe asthma. METHODS: The study first looked at potential targets for oxazolidine derivatives using SWISS-ADME. After the synthesis of the compounds, cytotoxicity and cytokine levels were analyzed. RESULTS: We demonstrated that LPSF/NB-12 and -13 reduced IFN-γ and IL-17 production in peripheral blood mononucleated cells from asthmatic patients in a concentrated manner. Our in silico analysis showed the neurokinin-1 receptor as a common target for both compounds, which is responsible for diverse proinflammatory effects of moderate and severe asthma. CONCLUSION: The work demonstrated a novel approach against asthma, which deserves further studies of its mechanisms of action.

Increases in [IP3]i aggravates diastolic [Ca2+] and contractile dysfunction in Chagas' human cardiomyocytes.

Chagas cardiomyopathy is the most severe manifestation of human Chagas disease and represents the major cause of morbidity and mortality in Latin America. We previously demonstrated diastolic Ca2+ alterations in cardiomyocytes isolated from Chagas' patients to different degrees of cardiac dysfunction. In addition, we have found a significant elevation of diastolic [Na+]d in Chagas' cardiomyocytes (FCII>FCI) that was greater than control. Exposure of cardiomyocytes to agents that enhance inositol 1,4,5 trisphosphate (IP3) generation or concentration like endothelin (ET-1) or bradykinin (BK), or membrane-permeant myoinositol 1,4,5-trisphosphate hexakis(butyryloxy-methyl) esters (IP3BM) caused an elevation in diastolic [Ca2+] ([Ca2+]d) that was always greater in cardiomyocytes from Chagas' than non- Chagas' subjects, and the magnitude of the [Ca2+]d elevation in Chagas' cardiomyocytes was related to the degree of cardiac dysfunction. Incubation with xestospongin-C (Xest-C), a membrane-permeable selective blocker of the IP3 receptors (IP3Rs), significantly reduced [Ca2+]d in Chagas' cardiomyocytes but did not have a significant effect on non-Chagas' cells. The effects of ET-1, BK, and IP3BM on [Ca2+]d were not modified by the removal of extracellular [Ca2+]e. Furthermore, cardiomyocytes from Chagas' patients had a significant decrease in the sarcoplasmic reticulum (SR) Ca2+content compared to control (Control>FCI>FCII), a higher intracellular IP3 concentration ([IP3]i) and markedly depressed contractile properties compared to control cardiomyocytes. These results provide additional and convincing support about the implications of IP3 in the pathogenesis of Chagas cardiomyopathy in patients at different stages of chronic infection. Additionally, these findings open the door for novel therapeutic strategies oriented to improve cardiac function and quality of life of individuals suffering from chronic Chagas cardiomyopathy (CC).

Effect of mate tea (Ilex paraguariensis) on the expression of the leukocyte NADPH oxidase subunit p47phox and on circulating inflammatory cytokines in healthy men: a pilot study.

Increased superoxide production by phagocytic NADPH oxidase has been associated with inflammatory conditions. Growing evidences suggest that dietary polyphenols may modulate the expression of NADPH oxidase subunits. Herein, we examined whether soluble mate tea (SMT) consumption - a polyphenol-rich beverage - affects the expression of the leukocyte NADPH oxidase protein p47phox and/or circulating biomarkers of inflammation and antioxidant biomarkers in humans. In a two-phase study, nine men were requested to drink water (control) for 8 d and then follow a second 8-d period drinking SMT. Blood samples were analysed for p47phox protein in CD16+/CD14- cells, interleukin (IL)-1ß (IL-1ß), tumour necrosis factor-alpha (TNF-α), IL-6, total phenols, and reduced and oxidised glutathione (GSH and GSSG, respectively) after each study phase. After SMT intake, CD16+/CD14- cells' p47phox protein and serum TNF-α and IL-6 levels were significantly attenuated (P < .05) while plasma phenolic compounds and blood GSH:GSSG ratio were significantly enhanced (P < .05). Consumption of SMT favourably affected leukocytes' p47phox expression and inflammatory cytokine and antioxidants levels in peripheral blood, which may help decrease oxidative stress and low-grade inflammation.

Analysis of 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutylanilide (DTMBA) as a new potential biomarker of exposure to vinclozolin in urine.

Vinclozolin (V) is a fungicide with anti-androgenic properties whose metabolism is not fully understood, and data on urinary elimination of either V or its metabolites are limited. Therefore the kinetics of urinary elimination of V and its metabolites, after an oral dose in adult male rats were investigated. A single oral dose of V (100 mg/kg) suspended in corn oil was administered to male adult Wistar rats, and urine was collected at different times after dosing. V and its metabolites were extracted from urine, then enzymatically hydrolyzed using ß-glucuronidase/sulfatase of H. pomatia, and analyzed by HPLC/DAD. Urinary pharmacokinetic parameters were calculated using the analyte concentrations adjusted by creatinine levels. V and its metabolites 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutylanilide (DTMBA, formerly denoted as M5), 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1), 3,5-dichloroaniline (M3), and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2) were efficiently detected. The mean urine concentrations of V and M1 metabolite were fitted to a two-compartmental model for pharmacokinetic analysis. DTMBA approximately represented 88% of the total excreted metabolites, it was easily detected up to 168 h after dosing and its half-lives were 21.5 and 74.1 h, respectively. M1 was the second most abundant metabolite and was detected up to 144 h after being void. V and M3 were detected before 48 h, and M2 exhibited the lowest levels during the first 8 h after dosing. DTMBA, the most abundant V metabolite is quickly eliminated by urine, it is chemically stable, specific and could represent a useful alternative to be used as a biomarker of exposure to V.

Novel insights into the metabolic pathway of iprodione by soil bacteria.

Microbial degradation constitutes the key soil dissipation process for iprodione. We recently isolated a consortium, composed of an Arthrobacter sp. strain C1 and an Achromobacter sp. strain C2, that was able to convert iprodione to 3,5-dichloroaniline (3,5-DCA). However, the formation of metabolic intermediates and the role of the strains on iprodione metabolism remain unknown. We examined the degradation of iprodione and its suspected metabolic intermediates, 3,5-dichlorophenyl-carboxamide (metabolite I) and 3,5-dichlorophenylurea-acetate (metabolite II), by strains C1 and C2 and their combination under selective (MSM) and nutrient-rich conditions (LB). Bacterial growth during degradation of the tested compounds was determined by qPCR. Strain C1 rapidly degraded iprodione (DT50 = 2.3 h) and metabolite II (DT50 = 2.9 h) in MSM suggesting utilization of isopropylamine, transiently formed by hydrolysis of iprodione, and glycine liberated during hydrolysis of metabolite II, as C and N sources. In contrast, strain C1 degraded metabolite I only in LB and growth kinetics suggested the involvement of a detoxification process. Strain C2 was able to transform iprodione and its metabolites only in LB. Strain C1 degraded vinclozolin, a structural analog of iprodione, and partially propanil, but not procymidone and phenylureas indicating a structure-dependent specificity related to the substituents of the carboxamide moiety.

Isolation of an aryloxyphenoxy propanoate (AOPP) herbicide-degrading strain Rhodococcus ruber JPL-2 and the cloning of a novel carboxylesterase gene (feh).

The strain JPL-2, capable of degrading fenoxaprop-P-ethyl (FE), was isolated from the soil of a wheat field and identified as Rhodococcus ruber. This strain could utilize FE as its sole carbon source and degrade 94.6% of 100 mg L(-1) FE in 54 h. Strain JPL-2 could also degrade other aryloxyphenoxy propanoate (AOPP) herbicides. The initial step of the degradation pathway is to hydrolyze the carboxylic acid ester bond. A novel esterase gene feh, encoding the FE-hydrolyzing carboxylesterase (FeH) responsible for this initial step, was cloned from strain JPL-2. Its molecular mass was approximately 39 kDa, and the catalytic efficiency of FeH followed the order of FE > quizalofop-P-ethyl > clodinafop-propargyl > cyhalofop-butyl > fluazifop-P-butyl > haloxyfop-P-methyl > diclofop-methy, which indicated that the chain length of the alcohol moiety strongly affected the hydrolysis activity of the FeH toward AOPP herbicides.

Isolation of an aryloxyphenoxy propanoate (AOPP) herbicide-degrading strain Rhodococcus ruber JPL-2 and the cloning of a novel carboxylesterase gene (feh)

The strain JPL-2, capable of degrading fenoxaprop-P-ethyl (FE), was isolated from the soil of a wheat field and identified as Rhodococcus ruber. This strain could utilize FE as its sole carbon source and degrade 94.6% of 100 mg L−1 FE in 54 h. Strain JPL-2 could also degrade other aryloxyphenoxy propanoate (AOPP) herbicides. The initial step of the degradation pathway is to hydrolyze the carboxylic acid ester bond. A novel esterase gene feh, encoding the FE-hydrolyzing carboxylesterase (FeH) responsible for this initial step, was cloned from strain JPL-2. Its molecular mass was approximately 39 kDa, and the catalytic efficiency of FeH followed the order of FE > quizalofop-P-ethyl > clodinafop-propargyl > cyhalofop-butyl > fluazifop-P-butyl > haloxyfop-P-methyl > diclofop-methy, which indicated that the chain length of the alcohol moiety strongly affected the hydrolysis activity of the FeH toward AOPP herbicides.

Isolation of an aryloxyphenoxy propanoate (AOPP) herbicide-degrading strain Rhodococcus ruber JPL-2 and the cloning of a novel carboxylesterase gene (feh)

The strain JPL-2, capable of degrading fenoxaprop-P-ethyl (FE), was isolated from the soil of a wheat field and identified as Rhodococcus ruber. This strain could utilize FE as its sole carbon source and degrade 94.6% of 100 mg L−1 FE in 54 h. Strain JPL-2 could also degrade other aryloxyphenoxy propanoate (AOPP) herbicides. The initial step of the degradation pathway is to hydrolyze the carboxylic acid ester bond. A novel esterase gene feh, encoding the FE-hydrolyzing carboxylesterase (FeH) responsible for this initial step, was cloned from strain JPL-2. Its molecular mass was approximately 39 kDa, and the catalytic efficiency of FeH followed the order of FE > quizalofop-P-ethyl > clodinafop-propargyl > cyhalofop-butyl > fluazifop-P-butyl > haloxyfop-P-methyl > diclofop-methy, which indicated that the chain length of the alcohol moiety strongly affected the hydrolysis activity of the FeH toward AOPP herbicides.(AU)

Enzyme molecular mechanism as a starting point to design new inhibitors: a theoretical study of O-GlcNAcase.

O-Glycoprotein 2-acetamino-2-deoxy-ß-D-glucopyranosidase (O-GlcNAcase) hydrolyzes O-linked 2-acetamido-2-deoxy-ß-D-glucopyranoside (O-GlcNAc) residues from post-translationally modified serine/threonine residues of nucleocytoplasmic protein. The chemical process involves substrate-assisted catalysis, where two aspartate residues have been identified as the two key catalytic residues of O-GlcNAcase. In this report, the first step of the catalytic mechanism used by O-GlcNAcase involving substrate-assisted catalysis has been studied using a hybrid quantum mechanical/molecular mechanical (QM/MM) Molecular Dynamics (MD) calculations. The free energy profile shows that the formation of the oxazoline intermediate in the O-GlcNAcase catalytic reaction takes place by means of a stepwise mechanism. The first step would be a cyclization of the acetomide group, which seems to be dependent on the proton transfer from a conserved aspartate, Asp298 in Clostridium perfringens O-GlcNAcase. From this new intermediate, a proton is transferred from the azoline ring to another conserved aspartate (Asp297) thus forming the oxazoline ion and departure of the aglycone. In addition, averaged values of protein-substrate interaction energy along the reaction path shows that, in fact, the transition states present the highest binding affinities. A deeper analysis of the binding contribution of the individual residues shows that Asp297, Asp298, and Asp401 are basically responsible of the stabilization of these complexes. These results would explain why O-(2-acetamido-2deoxy-d-glucopyranosylidene)amino-N-phenycarbamate (PUGNAc), 1,2-dideoxy-2'-methyl-α-D-glucopyranoso-[2,1-d]-Δ2'-thiazoline (NAG-thiazoline), and GlcNAcstatin derivatives are potent inhibitors of this enzyme, resembling the two transition states of the O-GlcNAcase catalytic reaction path. These results may be useful to rational design compounds with more interesting inhibitory activity.

IP(3)-dependent, post-tetanic calcium transients induced by electrostimulation of adult skeletal muscle fibers.

Tetanic electrical stimulation induces two separate calcium signals in rat skeletal myotubes, a fast one, dependent on Cav 1.1 or dihydropyridine receptors (DHPRs) and ryanodine receptors and related to contraction, and a slow signal, dependent on DHPR and inositol trisphosphate receptors (IP(3)Rs) and related to transcriptional events. We searched for slow calcium signals in adult muscle fibers using isolated adult flexor digitorum brevis fibers from 5-7-wk-old mice, loaded with fluo-3. When stimulated with trains of 0.3-ms pulses at various frequencies, cells responded with a fast calcium signal associated with muscle contraction, followed by a slower signal similar to one previously described in cultured myotubes. Nifedipine inhibited the slow signal more effectively than the fast one, suggesting a role for DHPR in its onset. The IP(3)R inhibitors Xestospongin B or C (5 µM) also inhibited it. The amplitude of post-tetanic calcium transients depends on both tetanus frequency and duration, having a maximum at 10-20 Hz. At this stimulation frequency, an increase of the slow isoform of troponin I mRNA was detected, while the fast isoform of this gene was inhibited. All three IP(3)R isoforms were present in adult muscle. IP(3)R-1 was differentially expressed in different types of muscle fibers, being higher in a subset of fast-type fibers. Interestingly, isolated fibers from the slow soleus muscle did not reveal the slow calcium signal induced by electrical stimulus. These results support the idea that IP(3)R-dependent slow calcium signals may be characteristic of distinct types of muscle fibers and may participate in the activation of specific transcriptional programs of slow and fast phenotype.