Relationships between microbial activity and soil physical and chemical properties in native and reforested Araucaria angustifolia forests in the state of São Paulo, Brazil

Araucaria angustifolia (Bert.) O. Kuntze is the main component of the Mixed Ombrophilous forest and, in the State of São Paulo, it is associated with a high diversity of soil organisms, essential for the maintenance of soil quality, making the conservation of this ecosystem a major and pressing challenge. The objective of this study was to identify the physical and chemical properties that are most closely correlated with dehydrogenase enzyme activity, basal respiration and microbial biomass under native (NF) and replanted (RF) Araucaria angustifolia forests in three regions of the state of São Paulo, in winter and summer. The main differentiating factors between the areas were also determined. Each forest was represented by three true replications; at each site, from around the araucaria trees, 15 soil samples (0-20 cm) were collected to evaluate the soil physical, chemical and microbiological properties. At the same points, forest litter was sampled to assess mass and chemical properties. The following microbiological properties were evaluated: microbial biomass carbon (MBC), basal respiration (CO2-C), metabolic quotient (Q: CO2), dehydrogenase enzyme activity (DHA) as well as the physical properties (moisture, bulk density, macroporosity and total porosity), soil chemical properties [pH, organic carbon (org-C), P, Ca, K, Mg, Al, H+Al], litter dry mass, and C, N and S contents. The data were subjected to analysis of variance (TWO-WAY: ANOVA). A Canonical Discriminant Analysis (CDA) and a Canonical Correlation Analysis (CCA) were also performed. In the soil under NF, the values of K, P, soil macroporosity, and litter dry mass were higher and Q: CO2 and DHA lower, regardless of the sampling period, and DHA was lower in winter. In the RF areas, the levels of moisture, porosity and Q: CO2 were higher in both sampling periods, and DHA was higher in winter. The MBC was only higher under NF in the summer, while the litter contents of C, N and S were greater in winter. In winter, CCA showed a high correlation of DHA with CO2-C, pH and H+Al, while in the summer org-C, moisture, Mg, pH and litter C were more associated with DHA and CO2-C. The CDA indicated H+Al, available P, total porosity, litter S content, and soil moisture as the most discriminating variables between NF and RF, but moisture was the most relevant, in both seasons and CO2-C only in winter. The combined analysis of CCA and CDA showed that the contribution of the microbiological variables to a differentiation of the areas was small at both samplings, which may indicate that the period after reforestation was long enough to allow an almost complete recovery of the microbial activity.

Effect of potassium sources on the antioxidant activity of eggplant¹

Potassium participates in the essential processes in plant physiology, however, the effects of K sources on plant metabolism have been little studied. Also, in certain cases, K sources and concentrations may cause undesirable effects, e.g., soil salinization. The objective was to evaluate the effect of K sources and levels on the enzyme activity of the antioxidant system and protein content in eggplant (Solanum melongena L.) leaves and to determine the most suitable K sources for these physiological characteristics. The experiment was conducted in randomized blocks, in a 2 × 4 factorial design, consisting of two K sources (KCl and K2SO4) and rates (250, 500, 750, and 1000 kg ha-1 K2O), with four replications. The following variables were evaluated: plant height, number of leaves per plant, superoxide dismutase (SOD), catalase (CAT), and leaf protein content. There was an increase in CAT activity with increasing K levels until 30 days after transplanting (DAT), when K2SO4 was applied and until 60 DAT, when KCl was used; after this period, the enzyme activity decreased under both sources. The activity of SOD increased in the presence of KCl, but was reduced with the application of K2SO4. For both K sources, increasing rates reduced the protein content and number of leaves per plant, and this reduction was greater under KCl application. Thus it was concluded that KCl tends more strongly to salinize the soil than K2SO4. Both for KCl and for K2SO4, the increasing rates adversely affected the activities of CAT and SOD and the levels of leaf protein in eggplant. The potential of KCl to reduce the enzyme activity of SOD and CAT, leaf protein content and plant growth of eggplant was stronger than that of K2SO4.

Activity of beta-glucosidase and levels of isoflavone glucosides in soybean cultivars affected by the environment

The enzyme beta-glucosidase hydrolyses the isoflavone glucosides developing aglycones, which are compounds with anticancer effects, that are also related with the astringency observed in soybean flavor. Due to the importance of this enzyme, a study was carried out to determine beta-glucosidase activity in soybean (Glycine max (L.) Merrill) cultivars with different contents of isoflavone glucosides (enzyme substrate). The enzyme activity was determined in 51 soybean cultivars sowed in Londrina (latitude 23ºS), in Paraná State, Brazil, and in the cultivar IAS 5 from soybean production regions of different Brazilian states. Among the cultivars, a range of variability of 176.1 to 96.3 units of enzyme activity (cultivars IAC-2 and Embrapa 2, respectively) was observed. A significant variability among cultivars could suggest genetic differences. In the states of Rio Grande do Sul, Paraná and Mato Grosso do Sul, the cultivar IAS 5 presented similar average of beta-glucosidase activity: 132.1, 131.9 and 132.5 units, respectively. Among locations in the states, the cultivar IAS 5 presented a variability for enzyme activity from 138.8 to 124.8 units, which were statistically different. In spite of statistics, the numerical values were not too different to assume that environmental conditions affected enzyme activity. A non-significative correlation for isoflavone glucoside concentrations and enzyme activity was observed among cultivars.

Antioxidant compoundsand total antioxidant activity in fruits of acerola from cv. Flor Branca, Florida Sweet and BRS 366

Information on antioxidant properties at different ontological stages may help producers and food technologists to identify which cultivar and/or maturity stage are most adequate for their need, therefore this work aimed to study the changes in the antioxidant metabolism during acerola development. Fruit from cv. Flor Branca, BRS366 and Florida Sweet were harvested at different stages: immature green colored (I), physiologically mature with green color and maximum size (II), breaker (III) and full red ripe (IV). After harvest, fruits were selected, divided into four replications with 500 g each and evaluated regarding their titratable acidity, pH, soluble solids, total soluble sugar, vitamin C, polyphenol, anthocyanin, yellow flavonoid, total antioxidant activity and antioxidant enzyme activity. Anthocyanin and flavonoid were determined through LC-DAD-ESI/MS and all analysis followed a completely randomized factorial 3 x 4 design. Fruits of 'Florida Sweet' presented significantly higher soluble solids (9.46ºBrix). Vitamin C content decreased during ripening, but ripe 'BRS 366' fruits showed the greatest values (1363 mg.100 g-1) and highest TAA with 42.36 µM TEAC.g-1FW. Cyanidin 3-rhamnoside (520.76 mg.100 g-1 DM) and quercetin 3-rhamnoside (33.72 mg.100 g-1 DM) were the most abundant anthocyanin and yellow flavonoids found mainly in 'Flor Branca' fruit of acerola, whose antioxidant enzymes activities were also higher. Ripe 'Florida Sweet' fruit presents a great potential for fresh consumption, meanwhile physiologically mature 'BRS 366' fruit seems the best option for the bioactive compounds processing industry. As 'Flor Branca' fruit of acerola kept the highest activity levels, it could be an indicative of greater potential for postharvest conservation.

Enzymatic activity of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase produced by Gordonia polyisoprenivorans

This study aimed to evaluate the environmental conditions for enzyme activity of catechol 1,2-dioxygenase (C1,2O) and catechol 2,3-dioxygenase (C2,3O) produced by Gordonia polyisoprenivorans in cell-free and immobilized extracts. The optimum conditions of pH, temperature, time course and effect of ions for enzyme activity were determined. Peak activity of C1,2O occurred at pH 8.0. The isolate exhibited the highest activity of C2,3O at pH 7.0 and 8.0 for the cell-free extract and immobilized extract, respectively. This isolate exhibited important characteristics such as broad range of pH, temperature and time course for enzyme activity.

Effect of phytoplasma infection on the activity of peroxidase, β-1,3 glucanase and chitinase in corn plants

In the present work we studied the effect of inoculating corn plants with the maize bushy stunt phytoplasma on the activity of the enzymes peroxidase, β-1,3 glucanase and chitinase. The experiments were carried out inside a greenhouse. Plants of a resistant and a susceptible corn hybrid were inoculated by using infective Dalbulus maidis leafhoppers 10 days after sowing. When symptoms started to appear, leaf samples were collected at different periods to quantify enzyme activity. The results showed an increase in the activity of the three enzymes in inoculated plants of both hybrids. In general, the values observed for the level of the different enzymes were higher in the susceptible hybrid when compared to the resistant one. Thus, the increases in peroxidase, β-1,3 glucanase and chitinase levels in inoculated plants are evidence of changes in the host metabolism caused by the phytoplasma. On the other hand, since the increases could not be correlated with plant resistance further studies are needed to explain such changes.

Acetolactate synthase activity in Euphorbia heterophylla resistant to ALS- and protox- inhibiting herbicides

The objective of this study was to determine the activity of the enzyme acetolactate synthase in biotypes of wild poinsettia (Euphorbia heterophylla) with multiple resistance to ALS- and Protox- inhibitors in the presence and absence of imazapyr, imazethapyr and nicosulfuron. We conducted in vitro assay of ALS enzyme extracted from plants of Vitorino, Bom Sucesso do Sul and Medianeira biotypes (with multiple resistance) and a susceptible population in the absence and presence of imazapyr, imazethapyr and nicosulfuron. In the absence of herbicides, biotypes with multiple resistance showed higher affinity for the substrate of the enzyme compared with the susceptible population. The herbicides imazapyr, imazethapyr and nicosulfuron had little effect on the enzyme activity of ALS-resistant biotypes and, conversely, high inhibitory effect on ALS of the susceptible population. Resistance factors were very high, greater than 438, 963 and 474 for Vitorino, Bom Sucesso do Sul and Medianeira biotypes, respectively. The resistance to ALS inhibitors is due to the insensitivity of ALS to herbicides of both imidazolinone and sulfonylurea groups, characterizing a cross-resistance.

Rapid eye movement (REM) sleep deprivation reduces rat frontal cortex acetylcholinesterase (EC 3.1.1.7) activity

Rapid eye movement (REM) sleep deprivation induces several behavioral changes. Among these, a decrease in yawning behavior produced by low doses of cholinergic agonists is observed which indicates a change in brain cholinergic neurotransmission after REM sleep deprivation. Acetylcholinesterase (Achase) controls acetylcholine (Ach) availability in the synaptic cleft. Therefore, altered Achase activity may lead to a change in Ach availability at the receptor level which, in turn, may result in modification of cholinergic neurotransmission. To determine if REM sleep deprivation would change the activity of Achase, male Wistar rats, 3 months old, weighing 250-300 g, were deprived of REM sleep for 96 h by the flower-pot technique (N = 12). Two additional groups, a home-cage control (N = 6) and a large platform control (N = 6), were also used. Achase was measured in the frontal cortex using two different methods to obtain the enzyme activity. One method consisted of the obtention of total (900 g supernatant), membrane-bound (100,000 g pellet) and soluble (100,000 g supernatant) Achase, and the other method consisted of the obtention of a fraction (40,000 g pellet) enriched in synaptic membrane-bound enzyme. In both preparations, REM sleep deprivation induced a significant decrease in rat frontal cortex Achase activity when compared to both home-cage and large platform controls. REM sleep deprivation induced a significant decrease of 16% in the membrane-bound Achase activity (nmol thiocholine formed min-1 mg protein-1) in the 100,000 g pellet enzyme preparation (home-cage group 152.1 ± 5.7, large platform group 152.7 ± 24.9 and REM sleep-deprived group 127.9 ± 13.8). There was no difference in the soluble enzyme activity. REM sleep deprivation also induced a significant decrease of 20% in the enriched synaptic membrane-bound Achase activity (home-cage group 126.4 ± 21.5, large platform group 127.8 ± 20.4, REM sleep-deprived group 102.8 ± 14.2). Our results suggest that REM sleep deprivation changes Ach availability at the level of its receptors through a decrease in Achase activity

Chronic imipramine treatment-induced changes in acetylcholinesterase (EC 3.1.1.7) activity in discrete rat brain regions

Cholinergic as well as monoaminergic neurotransmission seems to be involved in the etiology of affective disorders. Chronic treatment with imipramine, a classical antidepressant drug, induces adaptive changes in monoaminergic neurotransmission. In order to identify possible changes in cholinergic neurotransmission we measured total, membrane-bound and soluble acetylcholinesterase (Achase) activity in several rat brain regions after chronic imipramine treatment. Changes in Achase activity would indicate alterations in acetylcholine (Ach) availability to bind to its receptors in the synaptic cleft. Male rats were treated with imipramine (20 mg/kg, ip) for 21 days, once a day. Twenty-four hours after the last dose the rats were sacrificed and homogenates from several brain regions were prepared. Membrane-bound Achase activity (nmol thiocholine formed min-1 mg protein-1) after chronic imipramine treatment was significantly decreased in the hippocampus (control = 188.8 ± 19.4, imipramine = 154.4 ± 7.5, P<0.005) and striatum (control = 850.9 ± 59.6, imipramine = 742.5 ± 34.7, P<0.005). A small increase in total Achase activity was observed in the medulla oblongata and pons. No changes in enzyme activity were detected in the thalamus or total cerebral cortex. Since the levels of Achase seem to be enhanced through the interaction between Ach and its receptors, a decrease in Achase activity may indicate decreased Ach release by the nerve endings. Therefore, our data indicate that cholinergic neurotransmission is decreased after chronic imipramine treatment which is consistent with the idea of an interaction between monoaminergic and cholinergic neurotransmission in the antidepressant effect of imipramine

Constitutive nitric oxide synthase (cNOS) activity in Langerhans islets from streptozotocin diabetic rats

Nitric oxide synthase activity was measured in Langerhans islets isolated from control and streptozotocin diabetic rats. The activity of the enzyme was linear up to 150 µg of protein from control rats and was optimal at 0.1 µM calcium, when it was measured after 45 min of incubation at 37oC in the presence of 200 µM arginine. Specific activity of the enzyme was 25 x 10-4 nmol [3H]citrulline 45 min-1 mg protein-1. Streptozotocin diabetic rats exhibited less enzyme activity both in total pancreas homogenate and in isolated Langerhans islets when compared to control animals. Nitric oxide synthase activity measured in control and diabetic rats 15 days after the last streptozotocin injection in the second group of animals corresponded only to a constitutive enzyme since it was not inhibited by aminoguanidine in any of the mentioned groups. Hyperglycemia in diabetic rats may be the consequence of impaired insulin release caused at least in part by reduced positive modulation mediated by constitutive nitric oxide synthase activity, which was dramatically reduced in islets severely damaged after streptozotocin treatment.

Methylmercury intoxication and histochemical demonstration of NADPH-diaphorase activity in the striate cortex of adult cats

The effects of methylmercury (MeHg) on histochemical demonstration of the NADPH-diaphorase (NADPH-d) activity in the striate cortex were studied in 4 adult cats. Two animals were used as control. The contaminated animals received 50 ml milk containing 0.42 µg MeHg and 100 g fish containing 0.03 µg MeHg daily for 2 months. The level of MeHg in area 17 of intoxicated animals was 3.2 µg/g wet weight brain tissue. Two cats were perfused 24 h after the last dose (group 1) and the other animals were perfused 6 months later (group 2). After microtomy, sections were processed for NADPHd histochemistry procedures using the malic enzyme method. Dendritic branch counts were performed from camera lucida drawings for control and intoxicated animals (N = 80). Average, standard deviation and Student t-test were calculated for each data group. The concentrations of mercury (Hg) in milk, fish and brain tissue were measured by acid digestion of samples, followed by reduction of total Hg in the digested sample to metallic Hg using stannous chloride followed by atomic fluorescence analysis. Only group 2 revealed a reduction of the neuropil enzyme activity and morphometric analysis showed a reduction in dendritic field area and in the number of distal dendrite branches of the NADPHd neurons in the white matter (P<0.05). These results suggest that NADPHd neurons in the white matter are more vulnerable to the long-term effects of MeHg than NADPHd neurons in the gray matter.

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%.

A gasometric method to determine erythrocyte catalase activity

We describe a new gasometric method to determine erythrocyte catalase activity by the measurement of the volume of oxygen produced as a result of hydrogen peroxide decomposition in a system where enzyme and substrate are separated in a special reaction test tube connected to a manometer and the reagents are mixed with a motor-driven stirrer. The position of the reagents in the test tube permits the continuous measurement of oxygen evolution from the time of mixing, without the need to stop the reaction by the addition of acid after each incubation time. The enzyme activity is reported as KHb, i.e., mg hydrogen peroxide decomposed per second per gram of hemoglobin (s-1 g Hb-1). The value obtained for catalase activity in 28 samples of hemolyzed human blood was 94.4 ± 6.17 mg H2O2 s-1 g Hb-1. The results obtained were precise and consistent, indicating that this rapid, simple and inexpensive method could be useful for research and routine work.

Thyroid peroxidase activity is inhibited by amino acids

Normal in vitro thyroid peroxidase (TPO) iodide oxidation activity was completely inhibited by a hydrolyzed TPO preparation (0.15 mg/ml) or hydrolyzed bovine serum albumin (BSA, 0.2 mg/ml). A pancreatic hydrolysate of casein (trypticase peptone, 0.1 mg/ml) and some amino acids (cysteine, tryptophan and methionine, 50 µM each) also inhibited the TPO iodide oxidation reaction completely, whereas casamino acids (0.1 mg/ml), and tyrosine, phenylalanine and histidine (50 µM each) inhibited the TPO reaction by 54% or less. A pancreatic digest of gelatin (0.1 mg/ml) or any other amino acid (50 µM) tested did not significantly decrease TPO activity. The amino acids that impair iodide oxidation also inhibit the TPO albumin iodination activity. The inhibitory amino acids contain side chains with either sulfur atoms (cysteine and methionine) or aromatic rings (tyrosine, tryptophan, histidine and phenylalanine). Among the amino acids tested, only cysteine affected the TPO guaiacol oxidation reaction, producing a transient inhibition at 25 or 50 µM. The iodide oxidation inhibitory activity of cysteine, methionine and tryptophan was reversed by increasing iodide concentrations from 12 to 18 mM, while no such effect was observed when the cofactor (H2O2) concentration was increased. The inhibitory substances might interfere with the enzyme activity by competing with its normal substrates for their binding sites, binding to the free substrates or reducing their oxidized form.

Rapid eye movement sleep deprivation induces an increase in acetylcholinesterase activity in discrete rat brain regions

Some upper brainstem cholinergic neurons (pedunculopontine and laterodorsal tegmental nuclei) are involved in the generation of rapid eye movement (REM) sleep and project rostrally to the thalamus and caudally to the medulla oblongata. A previous report showed that 96 h of REM sleep deprivation in rats induced an increase in the activity of brainstem acetylcholinesterase (Achase), the enzyme which inactivates acetylcholine (Ach) in the synaptic cleft. There was no change in the enzyme's activity in the whole brain and cerebrum. The components of the cholinergic synaptic endings (for example, Achase) are not uniformly distributed throughout the discrete regions of the brain. In order to detect possible regional changes we measured Achase activity in several discrete rat brain regions (medulla oblongata, pons, thalamus, striatum, hippocampus and cerebral cortex) after 96 h of REM sleep deprivation. Naive adult male Wistar rats were deprived of REM sleep using the flower-pot technique, while control rats were left in their home cages. Total, membrane-bound and soluble Achase activities (nmol of thiocholine formed min-1 mg protein-1) were assayed photometrically. The results (mean ± SD) obtained showed a statistically significant (Student t-test) increase in total Achase activity in the pons (control: 147.8 ± 12.8, REM sleep-deprived: 169.3 ± 17.4, N = 6 for both groups, P<0.025) and thalamus (control: 167.4 ± 29.0, REM sleep-deprived: 191.9 ± 15.4, N = 6 for both groups, P<0.05). Increases in membrane-bound Achase activity in the pons (control: 171.0 ± 14.7, REM sleep-deprived: 189.5 ± 19.5, N = 6 for both groups, P<0.05) and soluble enzyme activity in the medulla oblongata (control: 147.6 ± 16.3, REM sleep-deprived: 163.8 ± 8.3, N = 6 for both groups, P<0.05) were also observed. There were no statistically significant differences in the enzyme's activity in the other brain regions assayed. The present findings show that the increase in Achase activity induced by REM sleep deprivation was specific to the pons, a brain region where cholinergic neurons involved in REM generation are located, and also to brain regions which receive cholinergic input from the pons (the thalamus and medulla oblongata). During REM sleep extracellular levels of Ach are higher in the pons, medulla oblongata and thalamus. The increase in Achase activity in these brain areas after REM sleep deprivation suggests a higher rate of Ach turnover.