Seeds of species from the ‘caatinga’: proteins, oils and fatty acid contents

The seeds of 14 species from the ‘caatinga’, a dry forest ecosystem of the semiarid region of northeast Brazil, were analysed for total protein and total lipid contents, as well as fatty acid distribution. The seeds of Argemone mexicana L., an introduced and naturalized species in Brazil, commonly found in ‘caatingas’ and other vegetation, were also analysed. The protein contents ranged from 123 g.kg-1 to 551 g.kg-1, higher contents being found in species of Leguminosae, but also in Jatropha mollissima (Pohl) Baill. (Euphorbiaceae, 409 g.kg-1). Oil contents ranged from 10 g.kg-1 to 400 g.kg-1. The contents of protein and oil were found to be inversely proportional in the seeds of most species, the figures for proteins being generally higher than those of oils. Most species presented either oleic or linoleic as predominant fatty acids. Cardiospermum cf. corindum L. presented eicosenoic acid as the predominant fatty acid.

Effect of abscisic acid on the mobilisation of galactomannan and embryo development of Sesbania virgata (Cav.) Pers. (Leguminosae - Faboideae)

Galactomannans (GM) are storage cell wall polysaccharides present in endospermic seeds of legumes. They are thought to be storage polymers, since it has been observed for a few species (among them Sesbania virgata) that they are completely broken down after germination and their products are transferred to the growing embryo. We examined the effect of 10-4 M abscisic acid (ABA) on the degradation of galactomannan in isolated endosperms and intact seeds of S. virgata. We found that after seed germination the initial embryo growth was retarded. Ultrastructural analysis showed that the embryo is completely surrounded by an endosperm which displays very thick galactomannan-containing cell walls. Although an inhibitory effect has been observed on the increase of fresh mass of the embryo, the effect of ABA on the dry mass was weaker and transitory (from 48 to 96 h). Endosperm dry mass and galactomannan degradation were significantly inhibited and the activity of alpha-galactosidase was strongly affected. The addition of ABA before and/or after the start of mobilisation in intact seeds or isolated endosperms, showed that whereas addition before mobilisation did not affect dry mass decrease in intact seeds, it was strongly affected in isolated endosperms. On the other hand, whereas it affected embryo fresh mass increase in intact seeds, but not in isolated embryos, no significant effect was observed on dry mass. These results suggest that ABA affects galactomannan degradation and by doing so, prevents water absorption by the embryo, rather than affect its dry mass. As ABA has been detected in the endosperm of seeds of S. virgata, it is proposed that it probably acts as a modulator of galactomannan mobilisation and consequently synchronises it with early growth of the embryo.

Treatment of Fe-containing Acid Mine Waters in Fixed Bed Adsorption Column with Calcite Side-Stone

The acid mining drainage is considered the most significant environmental pollution problem around the world for the extensive formation acidic leachates containing heavy metals. Adsorption is widely used methods in water treatment due to it easy operation and the availability of a wide variety of commercial adsorbent (low cost). The primary goal of this thesis was to investigate the efficiency of neutralizing agents, CaCO3 and CaSiO3, and metal adsorption materials with unmodified limestone from Company Nordkalk Oy. In addition to this, the side materials of limestone mining were tested for iron adsorption from acidic model solution. This study was executed at Lappeenranta University of Technology, Finland. The work utilised fixed-bed adsorption column as the main equipment and large fluidized column. Atomic absorption spectroscopy (AAS) and x-ray diffraction (XRD) was used to determine ferric removal and the composition of material respectively. The results suggest a high potential for the studied materials to be used a low cost adsorbents in acid mine drainage treatment. From the two studied adsorbents, the FS material was more suitable than the Gotland material. Based on the findings, it is recommended that further studies might include detailed analysis of Gotland materials.

Evaluation of the addition of ascorbic acid to the ration of cultivated Piaractus mesopotamicus (Characidae) on the infrapopulation of Anacanthorus penilabiatus (Monogenea)

Sixty Piaractus mesopotamicus Holmberg, 1887 (pacu) fry fed a diet containing 0, 50, 100 and 200 mg ascorbic acid/kg dry feed were studied to evaluate the effect on parasitic infestation by the monogenean Anacanthorus penilabiatus Boeger, Husak and Martins, 1995 (Monogenea: Dactylogyridae) for a period of 24 weeks. The temperature of the aquaria was measured daily and remained between 28 and 31oC. At the beginning of the experiment, fish showed 6.15 ± 0.33 cm standard length and 8.64 ± 1.62 g average body weight. A sample of fish was examined and showed 43 ± 17 monogeneans per fish. At the end of the experiment, the gills of control and vitamin C-treated fish were collected for parasite counts. Control fish had 42.5 parasites per fish, a significantly higher number (P<0.05) when compared with fish fed vitamin C, that showed 16.5 parasites per fish. Ascorbic acid fortification in the food promoted an increase in fish resistance to parasites. It is suggested that an optimum level of 139 mg/kg vitamin C supplementation either elicited better nutritional conditions by stimulating the appetite of the fish or improved the immune response.

Flavianate, an amino acid precipitant, is a competitive inhibitor of trypsin at pH 3.0

Textile dyes bind to proteins leading to selective co-precipitation of a complex involving one protein molecule and more than one dye molecule of opposite charge in acid solutions, in a process of reversible denaturation that can be utilized for protein fractionation. In order to understand what occurs before the co-precipitation, a kinetic study using bovine ß-trypsin and sodium flavianate was carried out based on reaction progress curve techniques. The experiments were carried out using a-CBZ-L-Lys-p-nitrophenyl ester as substrate which was added to 50 mM sodium citrate buffer, pH 3.0, containing varying concentrations of ß-trypsin and dye. The reaction was recorded spectrophotometrically at 340 nm for 30 min, and the families of curves obtained were analyzed simultaneously by fitting integrated Michaelis-Menten equations. The dye used behaved as a competitive inhibitor of trypsin at pH 3.0, with Ki = 99 µM; kinetic parameters for the substrate hydrolysis were: Km = 32 µM, and kcat = 0.38/min. The competitive character of the inhibition suggests a specific binding of the first dye molecule to His-57, the only positively charged residue at the active site of the enzyme.

Respiratory effects of kynurenic acid microinjected into the ventromedullary surface of the rat

Several studies demonstrate that, within the ventral medullary surface (VMS), excitatory amino acids are necessary components of the neural circuits involved in the tonic and reflex control of respiration and circulation. In the present study we investigated the cardiorespiratory effects of unilateral microinjections of the broad spectrum glutamate antagonist kynurenic acid (2 nmol/200 nl) along the VMS of urethane-anesthetized rats. Within the VMS only one region was responsive to this drug. This area includes most of the intermediate respiratory area, partially overlapping the rostral ventrolateral medulla (IA/RVL). When microinjected into the IA/RVL, kynurenic acid produced a respiratory depression, without changes in mean arterial pressure or heart rate. The respiratory depression observed was characterized by a decrease in ventilation, tidal volume and mean inspiratory flow and an increase in respiratory frequency. Therefore, the observed respiratory depression was entirely due to a reduction in the inspiratory drive. Microinjections of vehicle (200 nl of saline) into this area produced no significant changes in breathing pattern, blood pressure or heart rate. Respiratory depression in response to the blockade of glutamatergic receptors inside the rostral VMS suggests that neurons at this site have an endogenous glutamatergic input controlling the respiratory cycle duration and the inspiratory drive transmission.

Effect of all-trans retinoic acid on newly diagnosed acute promyelocytic leukemia patients: results of a Brazilian center

Thirty-seven patients with acute promyelocytic leukemia (APL) were treated with all-trans retinoic acid (ATRA). Patients received 45 mg m-2 day-1 po of ATRA until complete remission (CR) was achieved, defined as: a) presence of less than 5% blasts in the bone marrow, with b) white blood cells >103/mm3, c) platelets >105/mm3 and d) hemoglobin concentration >8 g/dl, with no blood or platelet transfusions. Thirty-one (83.7%) patients achieved CR by day 50, and 75% of these before day 30. Correction of the coagulopathy, achieved between days 2 and 10 (mean, 3 days), was the first evidence of response to treatment. Only one patient had been previously treated with chemotherapy and three had the microgranular variant M3 form. Dryness of skin and mucosae was the most common side effect observed in 82% of the patients. Thrombosis, hepatotoxicity and retinoid acid syndrome (RAS) were observed in 7 (19%), 6 (16%) and 4 (11%) patients, respectively. Thirteen (35%) patients had to be submitted to chemotherapy due to hyperleukocytosis (above 40 x 103/mm3) and six of these presented with new signs of coagulopathy after chemotherapy. Four (11%) patients died secondarily to intracerebral hemorrhage (IH) and two (5.4%) dropped out of the protocol due to severe ATRA side effects (one RAS and one hepatotoxicity). RAS and IH were related strictly to hyperleukocytosis. The reduced use of platelets and fresh frozen plasma probably lowered the total cost of treatment. We conclude that ATRA is an effective agent for inducing complete remission in APL patients.

Sex-dependent differences in the activities of acetylsalicylic acid-esterases in mouse kidneys

Acetylsalicylic acid (ASA), the most used drug worldwide, is hydrolyzed to salicylic acid and acetate by esterases present in tissues of several species including humans. Sex differences in drug metabolism by rodent liver are documented in the literature. In this paper we report a difference in the activities of the esterases (ASA-esterase I and II) in the kidneys of male and female mice. In this species there is no difference between males and females in liver ASA-esterases (ASA-esterase I: males 38.5 ± 7.9 (N = 5) and females 31.6 ± 7.6 (N = 5) nmol of salicylic acid formed min-1 mg protein-1, P>0.05; ASA-esterase II: males 77.3 ± 17.4 (N = 5) and females 61.4 ± 15.1 (N = 5) nmol of salicylic acid formed min-1 mg protein-1, P>0.05). However, in the kidneys males presented a much higher enzyme activity than females (ASA-esterase I: males 25.2 ± 6.3 (N = 5) and females 6.8 ± 0.6 (N = 5) nmol of salicylic acid formed min-1 mg protein-1, P<0.0002; ASA-esterase II: males 79.8 ± 10.1 (N = 5) and females 13.0 ± 1.1 (N = 5) nmol of salicylic acid formed min-1 mg protein-1, P<0.0001). The difference between sexes observed in mouse kidneys could serve as a model to study the molecular basis of this sex difference and also to determine the possible involvement of pituitary and gonadal hormones in this difference in ASA-esterase activities since these hormones control the sex differences in rodent liver enzyme activity.

Important amino acid residues of potato plant uncoupling protein (StUCP)

Chemical modifications were used to identify some of the functionally important amino acid residues of the potato plant uncoupling protein (StUCP). The proton-dependent swelling of potato mitochondria in K+-acetate in the presence of linoleic acid and valinomycin was inhibited by mersalyl (Ki = 5 µM) and other hydrophilic SH reagents such as Thiolyte MB, iodoacetate and 5,5'-dithio-bis-(2-nitrobenzoate), but not by hydrophobic N-ethylmaleimide. This pattern of inhibition by SH reagents was similar to that of brown adipose tissue uncoupling protein (UCP1). As with UCP1, the arginine reagent 2,3-butadione, but not N-ethylmaleimide or other hydrophobic SH reagents, prevented the inhibition of StUCP-mediated transport by ATP in isolated potato mitochondria or with reconstituted StUCP. The results indicate that the most reactive amino acid residues in UCP1 and StUCP are similar, with the exception of N-ethylmaleimide-reactive cysteines in the purine nucleotide-binding site.

Chronic postnatal administration of methylmalonic acid provokes a decrease of myelin content and ganglioside N-acetylneuraminic acid concentration in cerebrum of young rats

Levels of methylmalonic acid (MMA) comparable to those of human methylmalonic acidemia were achieved in blood (2-2.5 mmol/l) and brain (1.35 µmol/g) of rats by administering buffered MMA, pH 7.4, subcutaneously twice a day from the 5th to the 28th day of life. MMA doses ranged from 0.76 to 1.67 µmol/g as a function of animal age. Control rats were treated with saline in the same volumes. The animals were sacrificed by decapitation on the 28th day of age. Blood was taken and the brain was rapidly removed. Medulla, pons, the olfactory lobes and cerebellum were discarded and the rest of the brain ("cerebrum") was isolated. Body and "cerebrum" weight were measured, as well as the cholesterol and triglyceride concentrations in blood and the content of myelin, total lipids, and the concentrations of the lipid fractions (cholesterol, glycerolipids, phospholipids and ganglioside N-acetylneuraminic acid (ganglioside-NANA)) in the "cerebrum". Chronic MMA administration had no effect on body or "cerebrum" weight, suggesting that the metabolites per se neither affect the appetite of the rats nor cause malnutrition. In contrast, MMA caused a significant reduction of plasma triglycerides, but not of plasma cholesterol levels. A significant diminution of myelin content and of ganglioside-NANA concentration was also observed in the "cerebrum". We propose that the reduction of myelin content and ganglioside-NANA caused by MMA may be related to the delayed myelination/cerebral atrophy and neurological dysfunction found in methylmalonic acidemic children.

The mechanism of gentisic acid-induced relaxation of the guinea pig isolated trachea: the role of potassium channels and vasoactive intestinal peptide receptors

We examined some of the mechanisms by which the aspirin metabolite and the naturally occurring metabolite gentisic acid induced relaxation of the guinea pig trachea in vitro. In preparations with or without epithelium and contracted by histamine, gentisic acid caused concentration-dependent and reproducible relaxation, with mean EC50 values of 18 µM and Emax of 100% (N = 10) or 20 µM and Emax of 92% (N = 10), respectively. The relaxation caused by gentisic acid was of slow onset in comparison to that caused by norepinephrine, theophylline or vasoactive intestinal peptide (VIP). The relative rank order of potency was: salbutamol 7.9 > VIP 7.0 > gentisic acid 4.7 > theophylline 3.7. Gentisic acid-induced relaxation was markedly reduced (24 ± 7.0, 43 ± 3.9 and 78 ± 5.6%) in preparations with elevated potassium concentration in the medium (20, 40 or 80 mM, respectively). Tetraethylammonium (100 µM), a nonselective blocker of the potassium channels, partially inhibited the relaxation response to gentisic acid, while 4-AP (10 µM), a blocker of the voltage potassium channel, inhibited gentisic acid-induced relaxation by 41 ± 12%. Glibenclamide (1 or 3 µM), at a concentration which markedly inhibited the relaxation induced by the opener of ATP-sensitive K+ channels, levcromakalim, had no effect on the relaxation induced by gentisic acid. Charybdotoxin (0.1 or 0.3 µM), a selective blocker of the large-conductance Ca2+-activated K+ channels, caused rightward shifts (6- and 7-fold) of the gentisic acid concentration-relaxation curve. L-N G-nitroarginine (100 µM), a NO synthase inhibitor, had no effect on the relaxant effect of gentisic acid, and caused a slight displacement to the right in the relaxant effect of the gentisic acid curve at 300 µM, while methylene blue (10 or 30 µM) or ODQ (1 µM), the inhibitors of soluble guanylate cyclase, all failed to affect gentisic acid-induced relaxation. D-P-Cl-Phe6,Leu17[VIP] (0.1 µM), a VIP receptor antagonist, significantly inhibited (37 ± 7%) relaxation induced by gentisic acid, whereas CGRP (8-37) (0.1 µM), a CGRP antagonist, only slightly enhanced the action of gentisic acid. Taken together, these results provide functional evidence for the direct activation of voltage and large-conductance Ca+2-activated K+ channels, or indirect modulation of potassium channels induced by VIP receptors and accounts for the predominant relaxation response caused by gentisic acid in the guinea pig trachea.

Inhibition of in vitro CO2 production and lipid synthesis by 2-hydroxybutyric acid in rat brain

2-Hydroxybutyric acid appears at high concentrations in situations related to deficient energy metabolism (e.g., birth asphyxia) and also in inherited metabolic diseases affecting the central nervous system during neonatal development, such as "cerebral" lactic acidosis, glutaric aciduria type II, dihydrolipoyl dehydrogenase (E3) deficiency, and propionic acidemia. The present study was carried out to determine the effect of 2-hydroxybutyric acid at various concentrations (1-10 mM) on CO2 production and lipid synthesis from labeled substrates in cerebral cortex of 30-day-old Wistar rats in vitro. CO2 production was significantly inhibited (30-70%) by 2-hydroxybutyric acid in cerebral cortex prisms, in total homogenates and in the mitochondrial fraction. We also demonstrated a significant inhibition of lipid synthesis (20-45%) in cerebral cortex prisms and total homogenates in the presence of 2-hydroxybutyric acid. However, no inhibition of lipid synthesis occurred in homogenates free of nuclei and mitochondria. The results indicate an impairment of mitochondrial energy metabolism caused by 2-hydroxybutyric acid, a fact that may secondarily lead to reduction of lipid synthesis. It is possible that these findings may be associated with the neuropathophysiology of the situations where 2-hydroxybutyric acid is accumulated.

A time-dependent, two-step binding mode of the nitro dye flavianic acid to trypsin in acid media

Synthetic dyes bind to proteins causing selective coprecipitation of the complexes in acid aqueous solution by a process of reversible denaturation that can be used as an alternative method for protein fractionation. The events that occur before precipitation were investigated by equilibrium dialysis using bovine trypsin and flavianic acid as a model able to cause coprecipitation. A two-step mode of interaction was found to be dependent on the incubation periods allowed for binding, with pronounced binding occurring after 42 h of incubation. The first step seems to involve hydration effects and conformational changes induced by binding of the first dye molecule, following rapid denaturation due to the binding of six additional flavianate anions to the macromolecule.

Effect of L-arginine, dimercaptosuccinic acid (DMSA) and the association of L-arginine and DMSA on tissue lead mobilization and blood pressure level in plumbism

Lead (Pb)-induced hypertension is characterized by an increase in reactive oxygen species (ROS) and a decrease in nitric oxide (NO). In the present study we evaluated the effect of L-arginine (NO precursor), dimercaptosuccinic acid (DMSA, a chelating agent and ROS scavenger), and the association of L-arginine/DMSA on tissue Pb mobilization and blood pressure levels in plumbism. Tissue Pb levels and blood pressure evolution were evaluated in rats exposed to: 1) Pb (750 ppm, in drinking water, for 70 days), 2) Pb plus water for 30 more days, 3) Pb plus DMSA (50 mg kg-1 day-1, po), L-arginine (0.6%, in drinking water), and the combination of L-arginine/DMSA for 30 more days, and 4) their respective matching controls. Pb exposure increased Pb levels in the blood, liver, femur, kidney and aorta. Pb levels in tissues decreased after cessation of Pb administration, except in the aorta. These levels did not reach those observed in nonintoxicated rats. All treatments mobilized Pb from the kidney, femur and liver. Pb mobilization from the aorta was only effective with the L-arginine/DMSA treatment. Blood Pb concentrations in Pb-treated groups were not different from those of the Pb/water group. Pb increased blood pressure starting from the 5th week. L-arginine and DMSA treatments (4th week) and the combination of L-arginine/DMSA (3rd and 4th weeks) decreased blood pressure levels of intoxicated rats. These levels did not reach those of nonintoxicated rats. Treatment with L-arginine/DMSA was more effective than the isolated treatments in mobilizing Pb from tissues and in reducing the blood pressure of intoxicated rats.