Environmental and physiological factors that affect the efficacy of herbicides that inhibit the enzyme protoporphyrinogen oxidase: a literature review

Herbicides that inhibit the enzyme protoporphyrinogen oxidase (PROTOX) are usually effective to control dicotyledonous weeds and their agronomic efficacy is affected by environmental and physiological factors. The objective of this review is to summarize the knowledge of those factors available in the scientific literature in the last decade. Environmental factors that influence PROTOX inhibitors include temperature, irradiance and relative humidity. The most relevant physiological factors are the activity of enzymes that can detoxify herbicides and also of enzymes that mitigate the effects of oxidative stress in plants. The study also suggests some possible management strategies that could optimize the activity of PROTOX-inhibiting herbicides.

Extraction and Simultaneous Determination of Glyphosate, AMPA and Compounds of the Shikimic Acid Pathway in Plants

This study has aimed to develop a method for simultaneous extraction and determination by liquid chromatography and mass spectrometry (LC-MS/MS) of glyphosate, aminomethylphosphonic acid (AMPA), shikimic acid, quinic acid, phenylalanine, tyrosine and tryptophan. For the joint analysis of these compounds the best conditions of ionization in mass spectrometry and for chromatographic separation of the compounds were selected. Calibration curves and linearity ranges were also determined for each compound. Different extraction systems of the compounds were tested from plant tissues collected from sugarcane (Saccharum officinarum) and eucalyptus (Eucalyptus urophylla platiphylla) plants two days after the glyphosate application at the dose of 720 g a.e. ha-1. The plant material was dried in a forced air circulation drying oven and in a lyophilizer, and subsequently the extractions with acidified water (pH 2.5), acetonitrile-water (50:50) [v/v] and methanol-water (50:50) [v/v] were tested. To verify the recovery of the compounds in the plant matrix with acidified water as an extracting solution, the samples were fortified with a solution containing the mixture of the different analytical standards present so that this one presented the same levels of 50 and 100 μg L-1 of each compound. All experiments were conducted with three replicates. The analytical method developed was efficient for compounds quantifications. The extraction from the samples dried in an oven and using acidified water allowed better extraction levels for all compounds. The recovery levels of the compounds in the fortified samples with known amounts of each compound for both plants samples were rather satisfactory.

Characterization of nitrate reductase activity in vitro in Gracilaria caudata J. Agardh (Rhodophyta, Gracilariales)

The marine red alga Gracilaria caudata J. Agardh has been used in Brazil for agar extraction, mainly in the northeast region of the country. Nitrogen availability is the most important abiotic factor in seawater that limits the growth of seaweeds. The enzyme nitrate reductase (NR) is the key regulatory point in the nitrogen assimilation in photosynthetic organisms. This study describes an in vitro assay, characterizing the enzymatic activity of NR in terms of kinetic constants and stability, its oscillation during the day and glucose effect on NR modulation. Maximal peaks of NR activity were recorded at 20 ºC and pH 8.0. The enzymatic stability in crude extracts stored at 3 ± 1 ºC decreased significantly after 48 hours. Apparent Michaelis-Menten constants (K M) for NADH and nitrate were 22 µM and 3.95 mM, respectively. Gracilaria caudata NR activity showed an oscillation under light:dark photoperiod (14:10 hours LD) with 3-fold higher activity during the light phase, peaking after 10 hours of light. Under optimal assay conditions, the maximal activity was 92.9 10-3 U g-1. The addition of glucose induced the enzymatic activity during the light and dark phase, evidencing a possible modulation of this enzyme by the photosynthesis. This relationship can be explained by the need of carbon skeletons, produced by the photosynthetic process, to incorporate the intermediary metabolites of nitrate assimilatory pathway, avoiding the toxic intracellular accumulation of nitrite and ammonium. The optimization of enzymatic assay protocols for NR is essential to establish appropriate conditions to study nutritional behaviour, compare different taxonomic groups and to understand its regulatory mechanism.

Extraction of genomic DNA from Melipona quadrifasciata (Hymenoptera: Apidae, Meliponinae)

The objective of the present study was to test three different procedures for DNA extraction of Melipona quadrifasciata based on existing methods for DNA extraction of Apis, plants and fungi. These methods differ in the concentrations of specific substances in the extraction buffer. The results demonstrate that the method used for Apis is not adequate for DNA extraction from M. quadrifasciata. On the other hand, with minor modifications this method and the methods for plants and fungi were adequate for DNA extraction of this stingless bee, both for adults and larvae

Increased angiotensin-converting enzyme activity in the left ventricle after infarction

An increase in angiotensin-converting enzyme (ACE) activity has been observed in the heart after myocardial infarction (MI). Since most studies have been conducted in chronically infarcted individuals exhibiting variable degrees of heart failure, the present study was designed to determine ACE activity in an earlier phase of MI, before heart failure development. MI was produced in 3-month old male Wistar rats by ligation of the anterior branches of the left coronary artery, control rats underwent sham surgery and the animals were studied 7 or 15 days later. Hemodynamic data obtained for the anesthetized animals showed normal values of arterial blood pressure and of end-diastolic pressure in the right and left ventricular cavities of MI rats. Right and left ventricular (RV, LV) muscle and scar tissue homogenates were prepared to determine ACE activity in vitro by measuring the velocity of His-Leu release from the synthetic substrate Hyp-His-Leu. ACE activity was corrected to the tissue wet weight and is reported as nmol His-Leu g-1 min-1. No significant change in ACE activity in the RV homogenates was demonstrable. A small nonsignificant increase of ACE activity (11 &plusmn; 9%; P0.05) was observed 7 days after MI in the surviving left ventricular muscle. Two weeks after surgery, however, ACE activity was 46 &plusmn; 11% (P<0.05) higher in infarcted rats compared to sham-operated rats. The highest ACE activity was demonstrable in the scar tissue homogenate. In rats studied two weeks after surgery, ACE activity in the LV muscle increased from 105 &plusmn; 7 nmol His-Leu g-1 min-1 in control hearts to 153 &plusmn; 11 nmol His-Leu g-1 min-1 (P<0.05) in the remaining LV muscle of MI rats and to 1051 &plusmn; 208 nmol His-Leu g-1 min-1 (P<0.001) in the fibrous scar. These data indicate that ACE activity increased in the heart after infarction before heart failure was demonstrable by hemodynamic measurements. Since the blood vessels of the scar drain to the remaining LV myocardium, the high ACE activity present in the fibrous scar may increase the angiotensin II concentration and decrease bradykinin in the cardiac tissues surrounding the infarcted area. The increased angiotensin II in the fibrous scar may contribute to the reactive fibrosis and hypertrophy in the left ventricular muscle surviving infarction

Effect of protein malnutrition on the glycolytic and glutaminolytic enzyme activity of rat thymus and mesenteric lymph nodes

The activity of important glycolytic enzymes (hexokinase, phosphofructokinase, aldolase, phosphohexoseisomerase, pyruvate kinase and lactate dehydrogenase) and glutaminolytic enzymes (phosphate-dependent glutaminase) was determined in the thymus and mesenteric lymph nodes of Wistar rats submitted to protein malnutrition (6% protein in the diet rather than 20%) from conception to 12 weeks after birth. The wet weight (g) of the thymus and mesenteric lymph nodes decreased due to protein malnutrition by 87% (from 0.30 ± 0.05 to 0.04 ± 0.01) and 75% (0.40 ± 0.04 to 0.10 ± 0.02), respectively. The protein content was reduced only in the thymus from 102.3 ± 4.4 (control rats) to 72.6 ± 6.6 (malnourished rats). The glycolytic enzymes were not affected by protein malnutrition, but the glutaminase activity of the thymus and lymph nodes was reduced by half in protein-malnourished rats as compared to controls. This fact may lead to a decrease in the cellularity of the organ and thus in its size, weight and protein content.

Differential effects of isoproterenol on the activity of angiotensin-converting enzyme in the rat heart and aorta

The excessive stimulation of beta-adrenergic receptors in the heart induces myocardial hypertrophy. There are several experimental data suggesting that this hypertrophy may also depend, at least partially, on the increase of local production of angiotensin II secondary to the activation of the cardiac renin-angiotensin system. In this study we investigated the effects of isoproterenol on the activity of angiotensin-converting enzyme (ACE) in the heart and also in the aorta and plasma. Male Wistar rats weighing 250 to 305 g were treated with a dose of (±)-isoproterenol (0.3 mg kg-1 day-1, N = 8) sufficient to produce cardiac hypertrophy without deleterious effects on the pumping capacity of the heart. Control rats (N = 7) were treated with vehicle (corn oil). The animals were killed one week later. ACE activity was determined in vitro in the four cardiac chambers, aorta and plasma by a fluorimetric assay. A significant hypertrophy was observed in both ventricular chambers. ACE activity in the atria remained constant after isoproterenol treatment. There was a significant increase (P<0.05) of ACE activity in the right ventricle (6.9 ± 0.9 to 8.2 ± 0.6 nmol His-Leu g-1 min-1) and in the left ventricle (6.4 ± 1.1 to 8.9 ± 0.8 nmol His-Leu g-1 min-1). In the aorta, however, ACE activity decreased (P<0.01) after isoproterenol (41 ± 3 to 27 ± 2 nmol His-Leu g-1 min-1) while it remained unchanged in the plasma. These data suggest that ACE expression in the heart can be increased by stimulation of beta-adrenoceptors. However, this effect is not observed on other local renin-angiotensin systems, such as the aorta. Our data also suggest that the increased sympathetic discharge and the elevated plasma concentration of catecholamines may contribute to the upregulation of ACE expression in the heart after myocardial infarction and heart failure.

Hydrolysis of xylans by enzyme systems from solid cultures of Trichoderma harzianum strains

Xylanase activity was isolated from crude extracts of Trichoderma harzianum strains C and 4 grown at 28oC in a solid medium containing wheat bran as the carbon source. Enzyme activity was demonstrable in the permeate after ultrafiltration of the crude extracts using an Amicon system. The hydrolysis patterns of different xylans and paper pulps by xylanase activity ranged from xylose, xylobiose and xylotriose to higher xylooligosaccharides. A purified ß-xylosidase from the Trichoderma harzianum strain released xylose, xylobiose and xylotriose from seaweed, deacetylated, oat spelt and birchwood xylans. The purified enzyme was not active against acetylated xylan and catalyzed the hydrolysis of xylooligosaccharides, including xylotriose, xylotetraose and xylopentaose. However, the enzyme was not able to degrade xylohexaose. Xylanase pretreatment was effective for hardwood kraft pulp bleaching. Hardwood kraft pulp bleached in the XEOP sequence had its kappa number reduced from 13.2 to 8.9 and a viscosity of 20.45 cp. The efficiency of delignification was 33%.

Regulation of the expression of NADP-malic enzyme by UV-B, red and far-red light in maize seedlings

The induction of nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) in etiolated maize (Zea mays) seedlings by UV-B and UV-A radiation, and different levels of photosynthetically active radiation (PAR, 400-700 nm) was investigated by measuring changes in activity, protein quantity and RNA levels as a function of intensity and duration of exposure to the different radiations. Under low levels of PAR, exposure to UV-B radiation but not UV-A radiation for 6 to 24 h caused a marked increase in the enzyme levels similar to that observed under high PAR in the absence of UV-B. UV-B treatment of green leaves following a 12-h dark period also caused an increase in NADP-ME expression. Exposure to UV-B radiation for only 5 min resulted in a rapid increase of the enzyme, followed by a more gradual rise with longer exposure up to 6 h. Low levels of red light for 5 min or 6 h were also effective in inducing NADP-ME activity equivalent to that obtained with UV-B radiation. A 5-min exposure to far-red light following UV-B or red light treatment reversed the induction of NADP-ME, and this effect could be eliminated by further treatment with UV-B or red light. These results indicate that physiological levels of UV-B radiation can have a positive effect on the induction of this photosynthetic enzyme. The reducing power and pyruvate generated by the activity of NADP-ME may be used for respiration, in cellular repair processes and as substrates for fatty acid synthesis required for membrane repair.

Nonconventional amide bond formation catalysis: programming enzyme specificity with substrate mimetics

This article reports on the design and characteristics of substrate mimetics in protease-catalyzed reactions. Firstly, the basis of protease-catalyzed peptide synthesis and the general advantages of substrate mimetics over common acyl donor components are described. The binding behavior of these artificial substrates and the mechanism of catalysis are further discussed on the basis of hydrolysis, acyl transfer, protein-ligand docking, and molecular dynamics studies on the trypsin model. The general validity of the substrate mimetic concept is illustrated by the expansion of this strategy to trypsin-like, glutamic acid-specific, and hydrophobic amino acid-specific proteases. Finally, opportunities for the combination of the substrate mimetic strategy with the chemical solid-phase peptide synthesis and the use of substrate mimetics for non-peptide organic amide synthesis are presented.

Stimulatory effect of dexamethasone on angiotensin-converting enzyme in neonatal rat cardiac myocytes

Angiotensin-converting enzyme (ACE) plays a central role in cardiac remodeling associated with pathological conditions such as myocardial infarction. The existence of different cell types in the heart expressing components of the renin-angiotensin system makes it difficult to evaluate their relative role under physiological and pathological conditions. Since myocytes are the predominant cellular constituent of the heart by mass, in the present study we studied the effects of glucocorticoids on ACE activity using well-defined cultures of neonatal rat cardiac myocytes. Under steady-state conditions, ACE activity was present at very low levels, but after dexamethasone treatment ACE activity increased significantly (100 nmol/l after 24 h) in a time-dependent fashion. These results demonstrate the influence of dexamethasone on ACE activity in rat cardiac myocytes. This is consistent with the idea that ACE activation occurs under stress conditions, such as myocardial infarction, in which glucocorticoid levels may increase approximately 50-fold.

Standardization of a fluorimetric assay for the determination of tissue angiotensin-converting enzyme activity in rats

The tripeptide Hip-His-Leu was used to standardize a fluorimetric method to measure tissue angiotensin-converting enzyme (ACE) activity in rats. The fluorescence of the o-phthaldialdehyde-His-Leu adduct was compared in the presence and absence of the homogenate (25 µl) to determine whether the homogenate from different tissues interfered with the fluorimetric determination of the His-Leu product. Only homogenates from lung and renal medulla and cortex showed significantly altered fluorescence intensity. To overcome this problem, the homogenate from these tissues were diluted 10 times with assay buffer. The specificity of the assay was demonstrated by the inhibition of ACE activity with 3 µM enalaprilat (MK-422). There was a linear relationship between product formation and incubation time for up to 90 min for homogenates of renal cortex and medulla and liver, for up to 60 min for ventricles and adrenals and for up to 30 min for the aorta, lung and atrium homogenates. In addition, there was a linear relationship between product formation and the amount of protein in the homogenates within the following range: lung, 30-600 µg; renal cortex and medulla, 40-400 µg; atrium and ventricles, 20-200 µg; adrenal, 20-100 µg; aorta, 5-100 µg; liver, 5-25 µg. No peptidase activity against the His-Leu product (31 nmol), assayed in borate buffer (BB), was detected in the different homogenates except the liver homogenate, which was inhibited by 0.1 mM r-chloromercuribenzoic acid. ACE activity in BB was higher than in phosphate buffer (PB) due, at least in part, to a greater hydrolysis of the His-Leu product in PB. ACE activity of lung increased 20% when BB plus Triton was used. Enzyme activity was stable when the homogenates were stored at -20o or -70oC for at least 30 days. These results indicate a condition whereby ACE activity can be easily and efficiently assayed in rat tissue samples homogenized in BB using a fluorimetric method with Hip-His-Leu as a substrate.

Angiotensin-converting enzyme inhibition by lisinopril enhances liver regeneration in rats

Bradykinin has been reported to act as a growth factor for fibroblasts, mesangial cells and keratinocytes. Recently, we reported that bradykinin augments liver regeneration after partial hepatectomy in rats. Angiotensin-converting enzyme (ACE) is also a powerful bradykinin-degrading enzyme. We have investigated the effect of ACE inhibition by lisinopril on liver regeneration after partial hepatectomy. Adult male Wistar rats underwent 70% partial hepatectomy (PH). The animals received lisinopril at a dose of 1 mg kg body weight-1 day-1, or saline solution, intraperitoneally, for 5 days before hepatectomy, and daily after surgery. Four to six animals from the lisinopril and saline groups were sacrificed at 12, 24, 36, 48, 72, and 120 h after PH. Liver regeneration was evaluated by immunohistochemical staining for proliferating cell nuclear antigen using the PC-10 monoclonal antibody. The value for the lisinopril-treated group was three-fold above the corresponding control at 12 h after PH (P<0.001), remaining elevated at approximately two-fold above control values at 24, 36, 48 (P<0.001), and at 72 h (P<0.01) after PH, but values did not reach statistical difference at 120 h after PH. Plasma ACE activity measured by radioenzymatic assay was significantly higher in the saline group than in the lisinopril-treated group (P<0.001), with 81% ACE inhibition. The present study shows that plasma ACE inhibition enhances liver regeneration after PH in rats. Since it was reported that bradykinin also augments liver regeneration after PH, this may explain the liver growth stimulating effect of ACE inhibitors.

Regulation of antioxidant enzyme activities in male and female rat macrophages by sex steroids

Human and animal immune functions present sex dimorphism that seems to be mainly regulated by sex hormones. In the present study, the activities of the antioxidant enzymes total superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were measured in intraperitoneal resident macrophages from adult male and female rats. In addition to comparing males and females, we also examined the regulation of these enzyme activities in macrophages by sex steroids. GSH-Px activity did not differ between male and female macrophages. However, both total SOD and CAT activities were markedly higher in females than in males (83 and 180%). Removal of the gonads in both males and females (comparison between castrated groups) increased the difference in SOD activity from 83 to 138% and reduced the difference in CAT activity from 180 to 86%. Castration and testosterone administration did not significantly modify the activities of the antioxidant enzymes in male macrophages. Ovariectomy did not affect SOD or GSH-Px activity but markedly reduced (48%) CAT activity. This latter change was fully reversed by estrogen administration, whereas progesterone had a smaller effect. These results led us to conclude that differences in the SOD and CAT activities may partially explain some of the differences in immune function reported for males and females. Also, estrogen is a potent regulator of CAT in macrophages and therefore this enzyme activity in macrophages may vary considerably during the menstrual cycle.

Ectonucleotidase activities in Sertoli cells from immature rats

Sertoli cells have been shown to be targets for extracellular purines such as ATP and adenosine. These purines evoke responses in Sertoli cells through two subtypes of purinoreceptors, P2Y2 and P A1. The signals to purinoreceptors are usually terminated by the action of ectonucleotidases. To demonstrate these enzymatic activities, we cultured rat Sertoli cells for four days and then used them for different assays. ATP, ADP and AMP hydrolysis was estimated by measuring the Pi released using a colorimetric method. Adenosine deaminase activity (EC 3.5.4.4) was determined by HPLC. The cells were not disrupted after 40 min of incubation and the enzymatic activities were considered to be ectocellularly localized. ATP and ADP hydrolysis was markedly increased by the addition of divalent cations to the reaction medium. A competition plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. This result indicates that the enzyme that acts on the degradation of tri- and diphosphate nucleosides on the surface of Sertoli cells is a true ATP diphosphohydrolase (EC 3.6.1.5) (specific activities of 113 ± 6 and 21 ± 2 nmol Pi mg-1 min-1 for ATP and ADP, respectively). The ecto-5'-nucleotidase (EC 3.1.3.5) and ectoadenosine deaminase activities (specific activities of 32 ± 2 nmol Pi mg-1 min-1 for AMP and 1.52 ± 0.13 nmol adenosine mg-1 min-1, respectively) were shown to be able to terminate the effects of purines and may be relevant for the physiological control of extracellular levels of nucleotides and nucleosides inside the seminiferous tubules.