Evaluation of acid digestion techniques to estimate chromium contents in cattle feces

The objective of this work was to evaluate the accuracy of digestion techniques using nitric and perchloric acid at the ratios of 2:1, 3:1, and 4:1 v v-1, in one- or two-step digestion, to estimate chromium contents in cattle feces, using sodium molybdate as a catalyst. Fecal standards containing known chromium contents (0, 2, 4, 6, 8, and 10 g kg-1) were produced from feces of five animals. The chromium content in cattle feces is accurately estimated using digestion techniques based on nitric and perchloric acids, at a 3:1 v v-1 ratio, in one-step digestion, with sodium molybdate as a catalyst.

Use of sorghum straw (Sorghum bicolor) for second generation ethanol production: pretreatment and enzymatic hydrolysis

Agronomic biomass yields of forage sorghum BRS 655 presented similar results to other energy crops, producing 9 to 12.6 tons/ha (dry mass) of sorghum straw. The objective of this study was to evaluate the lignocellulosic part of this cultivar in terms of its potential in the different unit processes in the production of cellulosic ethanol, measuring the effects of pretreatment and enzymatic hydrolysis. Three types of pre-treatments for two reaction times were conducted to evaluate the characteristics of the pulp for subsequent saccharification. The pulp pretreated by alkali, and by acid followed by delignification, attained hydrolysis rates of over 90%.

Improved method to obtain pfaffic acid as a marker for quality control

Pfaffic acid, a marker of Hebanthe eriantha (Brazilian ginseng), was first isolated in 1983 but is not yet commercially available. This lack of availability compromises the quality control of this plant and its derivatives. This paper proposes a process for pfaffic acid isolation from roots of H. eriantha at a purity suitable for analytical purposes. The steps involved in this process included extraction, hydrolysis, fractionation and purification by preparative HPLC. This process led to isolation of pfaffic acid with a chromatographic purity of 98.5% in a 0.25% yield from dried roots of H. eriantha; this yield is more than forty times higher than that of the current method in the literature.

Assessment of NADPH-diaphorase stained myenteric neurons of the jejunum of diabetic rats supplemented with ascorbic acid

The relation between hyperglycemia and diabetic neuropathy has already been demonstrated in some studies. Among the theories proposed for its etiology the oxidative stress stands out. The performance of nitric oxide as a link between the metabolic and vascular neuropathogenic factors that triggers the diabetic neuropathy has already been put forward. This study aimed to assess the quantification and measurements of the cell body profile area (CBPA) of NADPH-diaphorase reactive (NADPH-dp) myenteric neurons of the jejunum of diabetic rats (induced by streptozotocin) supplemented with Ascorbic Acid (AA). These changes in the myenteric neurons seem to be related to the gastrointestinal disturbances observed in diabetes mellitus (DM). Twenty male Wistar rats (Rattus norvegicus) were distributed in 4 groups (n=5): controls (C), control supplemented (CS), diabetic (D), and diabetic suplemented (DS). DM was induced by estreptozotocin (50mg/kg body wt). One week after the induction and confirmation of the DM (glycemia exam), animals of the groups CS and DS received 50mg of AA three times a week by gavage. After 90 days of experiment, the animals were anesthetized with lethal thiopental dose (40mg/kg) and the collected jejunum processed for the histochemistry NADPH-diaphorase technique. Whole-mount preparations were obtained for quantitative and morphometric analysis of the myenteric neurons. A quantity of jejunum neurons in the Group D (96±7.5) was not different (P>0.05) from Group DS (116±8.08), C (92±9.7), and CS (81±5.4), but in Group DS the quantity was higher (P<0.05) than in Group C and CS. The CBPA of neurons from Group D (189.50±2.68µm²) and DS (195.92±3.75µm²) were lower (P<0.05) than from Group C (225.13±4.37µm²) and CS (210.23±3.15µm²). The streptozotocin-induced DM did not change the jejunum-ileum area, the jejunum myenteric plexus space organization and the density of NADPH-dp neurons. The 50g AA-supplementation, three times a week, during 90 days, did not decrease hyperglycemia; however, it had a neuroprotective effect on the myenteric neurons, minimizing the increase on the CBPA of NADPH-dp neurons and increasing the amount of NADPD-dp neurons.

Comparative uptake and metabolism of 2-[14C]-2,4-dichlorophenoxyacetic acid in callus cultures of monocot (Dioscorea spp.) and dicot (Nicotiana tabacum L.) plants

(Comparative uptake and metabolism of 2-[14C]-2,4-dichlorophenoxyacetic acid in callus cultures of monocot (Dioscorea spp.) and dicot (Nicotiana tabacum L.) plants). The uptake and metabolism of 2-[14C]-2,4-dichlorophenoxyacetic acid (2,4-D) were investigated in leaf calluses of Nicotiana tabacum, tuber calluses of Dioscorea opposita and calluses derived from zygotic embryos, leaves and petioles of Dioscorea composita. Striking similarities were evident in the patterns of 2,4-D metabolites and their chemical characteristics in the three callus types of D. composita compared, but significant differences were detected among the patterns of rnetabolites in the three species studied. Preliminary investigations on the stability of various metabolites (separated using TLC) by hydrolysis showed that sugar esters appeared to be the major metabolites in tobacco whilst in yams (D. opposita) glycosides were shown to be the main ones, which indicated a similarity between plants of Gramineae and Dioscoreaceae in terms of 2,4-D metabolism. Release of 2,4-D from tobacco callus cells upon their transfer to 2,4-D-free medium was detected and the implications of this are discussed in relation to the cultural conditions necessary to induce morphogenesis in vitro.

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.

Role of non-nitric oxide non-prostaglandin endothelium-derived relaxing factor(s) in bradykinin vasodilation

The most conspicuous effect of bradykinin following its administration into the systemic circulation is a transient hypotension due to vasodilation. In the present study most of the available evidence regarding the mechanisms involved in bradykinin-induced arterial vasodilation is reviewed. It has become firmly established that in most species vasodilation in response to bradykinin is mediated by the release of endothelial relaxing factors following the activation of B2-receptors. Although in some cases the action of bradykinin is entirely mediated by the endothelial release of nitric oxide (NO) and/or prostacyclin (PGI2), a large amount of evidence has been accumulated during the last 10 years indicating that a non-NO/PGI2 factor accounts for bradykinin-induced vasodilation in a wide variety of perfused vascular beds and isolated small arteries from several species including humans. Since the effect of the non-NO/PGI2 endothelium-derived relaxing factor is practically abolished by disrupting the K+ electrochemical gradient together with the fact that bradykinin causes endothelium-dependent hyperpolarization of vascular smooth muscle cells, the action of such factor has been attributed to the opening of K+ channels in these cells. The pharmacological characteristics of these channels are not uniform among the different blood vessels in which they have been examined. Although there is some evidence indicating a role for KCa or KV channels, our findings in the mesenteric bed together with other reports indicate that the K+ channels involved do not correspond exactly to any of those already described. In addition, the chemical identity of such hyperpolarizing factor is still a matter of controversy. The postulated main contenders are epoxyeicosatrienoic acids or endocannabinoid agonists for the CB1-receptors. Based on the available reports and on data from our laboratory in the rat mesenteric bed, we conclude that the NO/PGI2-independent endothelium-dependent vasodilation induced by BK is unlikely to involve a cytochrome P450 arachidonic acid metabolite or an endocannabinoid agonist.

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.

Arachidonic acid triggers an oxidative burst in leukocytes

The change in cellular reducing potential, most likely reflecting an oxidative burst, was investigated in arachidonic acid- (AA) stimulated leukocytes. The cells studied included the human leukemia cell lines HL-60 (undifferentiated and differentiated into macrophage-like and polymorphonuclear-like cells), Jurkat and Raji, and thymocytes and macrophages from rat primary cultures. The oxidative burst was assessed by nitroblue tetrazolium reduction. AA increased the oxidative burst until an optimum AA concentration was reached and the burst decreased thereafter. In the leukemia cell lines, optimum concentration ranged from 200 to 400 µM (up to 16-fold), whereas in rat cells it varied from 10 to 20 µM. Initial rates of superoxide generation were high, decreasing steadily and ceasing about 2 h post-treatment. The continuous presence of AA was not needed to stimulate superoxide generation. It seems that the NADPH oxidase system participates in AA-stimulated superoxide production in these cells since the oxidative burst was stimulated by NADPH and inhibited by N-ethylmaleimide, diphenyleneiodonium and superoxide dismutase. Some of the effects of AA on the oxidative burst may be due to its detergent action. There apparently was no contribution of other superoxide-generating systems such as xanthine-xanthine oxidase, cytochromes P-450 and mitochondrial electron transport chain, as assessed by the use of inhibitors. Eicosanoids and nitric oxide also do not seem to interfere with the AA-stimulated oxidative burst since there was no systematic effect of cyclooxygenase, lipoxygenase or nitric oxide synthase inhibitors, but lipid peroxides may play a role, as indicated by the inhibition of nitroblue tetrazolium reduction promoted by tocopherol.

Nitric oxide modulation of the hypothalamo-neurohypophyseal system

Nitric oxide (NO), a free radical gas produced endogenously from the amino acid L-arginine by NO synthase (NOS), has important functions in modulating vasopressin and oxytocin secretion from the hypothalamo-neurohypophyseal system. NO production is stimulated during increased functional activity of magnocellular neurons, in parallel with plastic changes of the supraoptic nucleus (SON) and paraventricular nucleus. Electrophysiological data recorded from the SON of hypothalamic slices indicate that NO inhibits firing of phasic and non-phasic neurons, while L-NAME, an NOS inhibitor, increases their activity. Results from measurement of neurohypophyseal hormones are more variable. Overall, however, it appears that NO, tonically produced in the forebrain, inhibits vasopressin and oxytocin secretion during normovolemic, isosmotic conditions. During osmotic stimulation, dehydration, hypovolemia and hemorrhage, as well as high plasma levels of angiotensin II, NO inhibition of vasopressin neurons is removed, while that of oxytocin neurons is enhanced. This produces a preferential release of vasopressin over oxytocin important for correction of fluid imbalance. During late pregnancy and throughout lactation, fluid homeostasis is altered and expression of NOS in the SON is down- and up-regulated, respectively, in parallel with plastic changes of the magnocellular system. NO inhibition of magnocellular neurons involves GABA and prostaglandin synthesis and the signal-transduction mechanism is independent of the cGMP-pathway. Plasma hormone levels are unaffected by icv 1H-[1, 2, 4]oxadiazolo-[4,3-a]quinoxalin-1-one (a soluble guanylyl cyclase inhibitor) or 8-Br-cGMP administered to conscious rats. Moreover, cGMP does not increase in homogenates of the neural lobe and in microdialysates of the SON when NO synthesis is enhanced during osmotic stimulation. Among alternative signal-transduction pathways, nitrosylation of target proteins affecting activity of ion channels is considered.

Effects of acid-base imbalance on vascular reactivity

Acid-base homeostasis maintains systemic arterial pH within a narrow range. Whereas the normal range of pH for clinical laboratories is 7.35-7.45, in vivo pH is maintained within a much narrower range. In clinical and experimental settings, blood pH can vary in response to respiratory or renal impairment. This altered pH promotes changes in vascular smooth muscle tone with impact on circulation and blood pressure control. Changes in pH can be divided into those occurring in the extracellular space (pHo) and those occurring within the intracellular space (pHi), although, extracellular and intracellular compartments influence each other. Consistent with the multiple events involved in the changes in tone produced by altered pHo, including type of vascular bed, several factors and mechanisms, in addition to hydrogen ion concentration, have been suggested to be involved. The scientific literature has many reports concerning acid-base balance and endothelium function, but these concepts are not clear about acid-base disorders and their relations with the three known mechanisms of endothelium-dependent vascular reactivity: nitric oxide (NO/cGMP-dependent), prostacyclin (PGI2/cAMP-dependent) and hyperpolarization. During the last decades, many studies have been published and have given rise to confronting data on acid-base disorder and endothelial function. Therefore, the main proposal of this review is to provide a critical analysis of the state of art and incentivate researchers to develop more studies about these issues.

Modulation of peritoneal macrophage activity by the saturation state of the fatty acid moiety of phosphatidylcholine

To determine the effects of saturated and unsaturated fatty acids in phosphatidylcholine (PC) on macrophage activity, peritoneal lavage cells were cultured in the presence of phosphatidylcholine rich in saturated or unsaturated fatty acids (sat PC and unsat PC, respectively), both used at concentrations of 32 and 64 µM. The treatment of peritoneal macrophages with 64 µM unsat PC increased the production of hydrogen peroxide by 48.3% compared to control (148.3 ± 16.3 vs 100.0 ± 1.8%, N = 15), and both doses of unsat PC increased adhesion capacity by nearly 50%. Moreover, 64 µM unsat PC decreased neutral red uptake by lysosomes by 32.5% compared to the untreated group (67.5 ± 6.8 vs 100.0 ± 5.5%, N = 15), while both 32 and 64 µM unsat PC decreased the production of lipopolysaccharide-elicited nitric oxide by 30.4% (13.5 ± 2.6 vs 19.4 ± 2.5 µM) and 46.4% (10.4 ± 3.1 vs 19.4 ± 2.5 µM), respectively. Unsat PC did not affect anion production in non-stimulated cells or phagocytosis of unopsonized zymosan particles. A different result pattern was obtained for macrophages treated with sat PC. Phorbol 12-miristate 13-acetate-elicited superoxide production and neutral red uptake were decreased by nearly 25% by 32 and 64 µM sat PC, respectively. Sat PC did not affect nitric oxide or hydrogen peroxide production, adhesion capacity or zymosan phagocytosis. Thus, PC modifies macrophage activity, but this effect depends on cell activation state, fatty acid saturation and esterification to PC molecule and PC concentration. Taken together, these results indicate that the fatty acid moiety of PC modulates macrophage activity and, consequently, is likely to affect immune system regulation in vivo.

5-HT1A autoreceptor modulation of locomotor activity induced by nitric oxide in the rat dorsal raphe nucleus

The dorsal raphe nucleus (DRN) is the origin of ascending serotonergic projections and is considered to be an important component of the brain circuit that mediates anxiety- and depression-related behaviors. A large fraction of DRN serotonin-positive neurons contain nitric oxide (NO). Disruption of NO-mediated neurotransmission in the DRN by NO synthase inhibitors produces anxiolytic- and antidepressant-like effects in rats and also induces nonspecific interference with locomotor activity. We investigated the involvement of the 5-HT1A autoreceptor in the locomotor effects induced by NO in the DRN of male Wistar rats (280-310 g, N = 9-10 per group). The NO donor 3-morpholinosylnomine hydrochloride (SIN-1, 150, and 300 nmol) and the NO scavenger S-3-carboxy-4-hydroxyphenylglycine (carboxy-PTIO, 0.1-3.0 nmol) were injected into the DRN of rats immediately before they were exposed to the open field for 10 min. To evaluate the involvement of the 5-HT1A receptor and the N-methyl-D-aspartate (NMDA) glutamate receptor in the locomotor effects of NO, animals were pretreated with the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 8 nmol), the 5-HT1A receptor antagonist N-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-N-2-pyridinyl-cyclohexanecarboxamide maleate (WAY-100635, 0.37 nmol), and the NMDA receptor antagonist DL-2-amino-7-phosphonoheptanoic acid (AP7, 1 nmol), followed by microinjection of SIN-1 into the DRN. SIN-1 increased the distance traveled (mean ± SEM) in the open-field test (4431 ± 306.1 cm; F7,63 = 2.44, P = 0.028) and this effect was blocked by previous 8-OH-DPAT (2885 ± 490.4 cm) or AP7 (3335 ± 283.5 cm) administration (P < 0.05, Duncan test). These results indicate that 5-HT1A receptor activation and/or facilitation of glutamate neurotransmission can modulate the locomotor effects induced by NO in the DRN.

Effects of sequential enzymatic hydrolysis on structural, bioactive and functional properties of Phaseolus lunatus protein isolate

Significant initiatives exist within the global food market to search for new, alternative protein sources with better technological, functional, and nutritional properties. Lima bean (Phaseolus lunatus L.) protein isolate was hydrolyzed using a sequential pepsin-pancreatin enzymatic system. Hydrolysis was performed to produce limited (LH) and extensive hydrolysate (EH), each with different degrees of hydrolysis (DH). The effects of hydrolysis were evaluated in vitro in both hydrolysates based on structural, functional and bioactive properties. Structural properties analyzed by electrophoretic profile indicated that LH showed residual structures very similar to protein isolate (PI), although composed of mixtures of polypeptides that increased hydrophobic surface and denaturation temperature. Functionality of LH was associated with amino acid composition and hydrophobic/hydrophilic balance, which increased solubility at values close to the isoelectric point. Foaming and emulsifying activity index values were also higher than those of PI. EH showed a structure composed of mixtures of polypeptides and peptides of low molecular weight, whose intrinsic hydrophobicity and amino acid profile values were associated with antioxidant capacity, as well as inhibiting angiotensin-converting enzyme. The results obtained indicated the potential of Phaseolus lunatus hydrolysates to be incorporated into foods to improve techno-functional properties and impart bioactive properties.

Screening and kinetics of glutaminase and glutamate decarboxylase producing lactic acid bacteria from fermented Thai foods

L-glutaminase and glutamic acid decarboxylase (GAD) catalyzes the hydrolysis of L-glutamine and glutamate, respectively. L-glutaminase widely used in cancer therapy along with a combination of other enzymes and most importantly these enzymes were used in food industries, as a major catalyst of bioconversion. The current investigation was aimed to screen and select L-glutaminase, and GAD producing lactic acid bacteria (LAB). A total of 338 LAB were isolated from fermented meat, fermented fish, fermented soya bean, fermented vegetables and fruits. Among 338 isolates, 22 and 237 LAB has been found to be positive for L-glutaminase and GAD, respectively. We found that 30 days of incubation at 35 ºC and pH 6.0 was the optimum condition for glutaminase activity by G507/1. G254/2 was found to be the best for GAD activity with the optimum condition of pH 6.5, temperature 40 ºC and ten days of incubation. These LAB strains, G507/1 and G254/2, were identified as close relative of Lactobacillus brevis ATCC 14869 and Lactobacillus fermentum NBRC 3956, respectively by 16S rRNA sequencing. Further, improvements in up-stream of the fermentation process with these LAB strains are currently under development.