Amperometric biosensor for ascorbic acid

A L-ascorbic acid biosensor based on ascorbate oxidase has been developed. The enzyme was extracted from the mesocarp of cucumber (Cucumis sativus) by using 0.05 mol L-1 phosphate buffer, pH 5.8 containing 0.5 mol L-1 NaCl. After the dialysis versus phosphate buffer 0.05 mol L-1 pH 5.8, the enzyme was immobilized onto nylon net through glutaraldehyde covalent bond. The membrane was coupled to an O2 electrode and the yielding reaction monitored by oxygen depletion at -600 mV using flow injection analysis optimized to 0.1 mol L-1 phosphate buffer pH 5.8, as the carrier solution and flow-rate of 0.5 mL min-1. The ascorbic acid calibration curve was linear from 1.2x10-4 to 1.0x10-3 mol L-1. The evaluation of biosensor lifetime leads to 500 injections. Commercial pharmaceutical samples were analyzed with the proposed method and the results were compared with those obtained by high-performance liquid chromatography (HPLC).

Establishment of anammox process in sludge samples collected from swine wastewater treatment system

The high load of nitrogen present in swine wastewater is one of the biggest management challenges of the activity. The Anammox process emerges as a good alternative for biological removal of nitrogen. This study aims to acclimate sludge collected from swine effluent treatment systems to establish the Anammox process. Two sludge samples were collected at Embrapa Swine and Poultry, Concordia - SC, Brazil, one from the bottom of an inactive anaerobic pond (inoculum A) and another from an aeration tank (inoculum B). Both were acclimated until the depletion of NO3-N, being subsequently inoculated in two reactors (Reactor A - Inoculum A and Reactor B - Inoculum B). The Reactor A showed activity after 110 days of operation, while the Reactor B needed 170 days. The difference in the start-up time could be explained by the different environmental conditions to which each sludge was submitted. FISH and PCR analyses confirmed the presence of microorganisms with Anammox activity, demonstrating that the sludge of swine wastewater treatment systems is a good source of inoculum for the development of the Anammox process.

Estenose arterial pós-transplante hepático: tratamento com angioplastia transluminal percutânea

Vascular complications after liver transplantation include oclusion or stenosis at the sites of anastomosis in the hepatic artery, portal vein, and vena cava. Balloon angioplasty of these stenosis carries little risk and is a useful procedure for the treatment of these problems. The purpose of this paper was to assess whether percutaneous transluminal angioplasty can help to prolong allograft survival and impruve allograft function in patient with hepatic artery stenosis after liver transplantation. We report a 43-year-old mate with stenosis of hepatic artery anastomosis after liver transplantation. An abrupt elevation of liver enzymes and serum bilirrubin levels was noted on the fifth postoperative month. The patient underwent percutaneous liver biopsy, which revealed important ductal depletion due to hypoperfusion, even though Doppler ultrasound examination demonstrated arterial flow. An angiogram confirmed severe stenosis of the arterial anastomosis with poor intraparenchymal arterial perfusion pattern. In an attempt to preserve the graft, a percutaneous transluminal angioplasty was performed using microballoons mounted on a hydrophylic micro guidewire. Intervention proceeded without complications. Liver enzimes and bilirrubin levels decreased within twenty-four hours of angioplasty. Normal levels were achieved after one week. Seven month after angioplasty, the patient is in a optimal clinical condition with no signs of graft impairment. We conclude that percutaneous transluminal angioplasty of hepatic artery stenosis after liver transplantation is relatively safe and may help decrease allograft loss.

Spontaneous poisoning by larvae of Perreyia flavipes (Pergidae) in sheep

From a flock of 175 Texel sheep 25 animals died after consumption of a sawfly larvae subsequently identified as Perreyia flavipes. The disease occurred in June-July 2006 on a farm located in the county of Encruzilhada do Sul, Rio Grande do Sul, Brazil. Although there were 11 cattle in the same paddock, none of them was affected. High numbers of compact masses containing up to 150 larvae were scattered in the paddock where the animals were grazing. Most affected sheep showed severe apathy during 24-36 h before death, but weakness, muscular tremors and depression were also observed. Necropsy was performed on six sheep and the main macroscopic lesions were hemorrhages in the subcutaneous tissues, endocardium, gallbladder wall, and abomasal mucosa. In all animals was found hydrothorax, hydropericardium, ascites, and mild jaundice. Edema in the abomasal folds, mesentery, perirenal tissues, and gallbladder wall were also seen. The livers were yellowish with disseminated pinpoint hemorrhages in the parenchyma and had an enhanced lobular pattern. Perreyia flavipes larval body fragments and heads were found in the forestomach contents of the six sheep. Feces were scant, dry and formed balls coated by mucus and streaks of blood. Similar contents were also present at the end of the cecum. Prominent microscopic lesions included severe and diffuse periacinar or massive necrosis of hepatocytes associated with multifocal random hemorrhages. Diffuse necrosis of lymphoid follicles in lymph nodes and Peyer's patches, lymphoid depletion and necrosis in germinative centers of the spleen, and diffuse vacuolization in the renal tubular epithelia were also seen.

Intestinal lesions in pigs affected with postweaning multisystemic wasting syndrome

Samples of mesenteric lymph nodes and intestines from 79 unthrifty 3- to 5-month-old postweaning pigs, confirmed as naturally affected with postweaning multisystemic wasting syndrome (PMWS), were studied. Pigs originated from 12 farms in southern Brazil and were selected on the basis of clinical signs and/or gross lesions suggestive of enteric disorder. Lymphohistiocytic infiltrates of varying intensity were associated with anti-porcine circovirus type 2 (anti-PCV2) immunostaining (IS) in samples of intestines and mesenteric lymph nodes from all pigs. Although most findings were similar to those described in PCV2-associated enteritis, anti-PCV2 IS in association with depletion of the goblet cell mucin stores (24 pigs), diffuse ileal villous atrophy and fusion (18 pigs), and dilatation of the lymphatic vessels (11 pigs) combined or not with lymphangitis were also observed. PCV2 antigen was immunohistochemically demonstrated in the cytoplasm and nuclei from intralesional epithelial cells, histiocytes, and endothelial-like cells in intestinal tissues. Together these findings imply an association with PCV2. The presence of co-infections by Lawsonia intracellularis, Brachyspira spp., Mycobacterium spp., Salmonella spp., rotavirus, parvovirus, coronavirus and enteric calicivirus with PCV2 in the intestinal lesions was investigated.

Metabolic evaluation of Crioulo horses participating in competitions of 750 km

The Crioulo breed of horses performs in one of the most physically demanding equestrian competitions, the Marcha de Resistência, which is a contest in which the horses run 750 km in 15 days. The study's aim was to characterize the metabolic responses during this period. We evaluated eleven Crioulo horses in the competition, specifically, two males and nine females. Blood samples were collected 24 hours before the contest and on the 4th, 9th, 11th, 14th and 15th days of competition. We evaluated CK, AST, LDH, glucose, lactate, urea, creatinine, sodium, potassium, chloride, magnesium, total calcium, ionized calcium, total protein, hematocrit and the white blood cell count. At the end of the competition, the mean values of serum AST were 1151±358 IU/ L the mean LDH values were 7418±1695 IU/L and CK was 13,867±3998UI /L. There was a significant increase in urea, creatinine and lactate (p<0.0001). A decrease in the mean values of chloride, sodium, potassium, and total and ionized calcium was observed (p≤0.0002). An evaluation of the total leukocytes and segmented neutrophils (p≤0.0002) revealed their increased values, and decreased values were observed for hematocrit, plasma protein and total lymphocytes (p≤0.0003). The values of glucose, on average, remained constant. Based on these data, we conclude that the Marcha de Resistência competition necessitated a high muscular demand and the depletion of energy and electrolytes, suggesting an inflammatory process in the animals evaluated.

Water use efficiency of cassava plants under competition conditions

The objective of this work was to evaluate characteristics associated with the photosynthetic activity of cassava plants in competition with weeds or not. The trial was performed on open environment conditions, with experimental units consisting of fiber glass vases with 150 dm³ filled with Red Yellow Latosol, previously fertilized. Treatments consisted in the cultivation of cassava plants isolated and associated to three weed species (Bidens pilosa, Commelina benghalensis and Brachiaria plantaginea). After cassava shooting, 15 days after planting, a removal of the weeds excess was performed, sown at the time of cassava planting, leaving six plants m-2 of B. pilosa and four plants m-2 of C. benghalensis and B. plantaginea. At 60 days after emergence (DAE), stomatal conductance (Gs), vapor pressure in the substomatal cavity (Ean), temperature gradient between leaf and air (ΔT), transpiration rate (E) and water use efficiency (WUE) were evaluated. B. pilosa showed greater capacity to affect growth of cassava plants. B. plantaginea is very efficient in using water, especially by presenting C4 metabolism, and remains competitive with cassava even under temporarily low water status. C. benghalensis, in turn, is not a good competitor for light and apparently is not the primary cause of water depletion in the soil. The effects of weeds, in this case, were more associated with the competition. However, they were found between moderate to low. This implies that the competition established at experimental level was low.

Ca2+ dependence of gluconeogenesis stimulation by glucagon at different cytosolic NAD+-NADH redox potentials

The influence of Ca2+ on hepatic gluconeogenesis was measured in the isolated perfused rat liver at different cytosolic NAD+-NADH potentials. Lactate and pyruvate were the gluconeogenic substrates and the cytosolic NAD+-NADH potentials were changed by varying the lactate to pyruvate ratios from 0.01 to 100. The following results were obtained: a) gluconeogenesis from lactate plus pyruvate was not affected by Ca2+-free perfusion (no Ca2+ in the perfusion fluid combined with previous depletion of the intracellular pools); gluconeogenesis was also poorly dependent on the lactate to pyruvate ratios in the range of 0.1 to 100; only for a ratio equal to 0.01 was a significantly smaller gluconeogenic activity observed in comparison to the other ratios. b) In the presence of Ca2+, the increase in oxygen uptake caused by the infusion of lactate plus pyruvate at a ratio equal to 10 was the most pronounced one; in Ca2+-free perfusion the increase in oxygen uptake caused by lactate plus pyruvate infusion tended to be higher for all lactate to pyruvate ratios; the most pronounced difference was observed for a lactate/pyruvate ratio equal to 1. c) In the presence of Ca2+ the effects of glucagon on gluconeogenesis showed a positive correlation with the lactate to pyruvate ratios; for a ratio equal to 0.01 no stimulation occurred, but in the 0.1 to 100 range stimulation increased progressively, producing a clear parabolic dependence between the effects of glucagon and the lactate to pyruvate ratio. d) In the absence of Ca2+ the relationship between the changes caused by glucagon in gluconeogenesis and the lactate to pyruvate ratio was substantially changed; the dependence curve was no longer parabolic but sigmoidal in shape with a plateau beginning at a lactate/pyruvate ratio equal to 1; there was inhibition at the lactate to pyruvate ratios of 0.01 and 0.1 and a constant stimulation starting with a ratio equal to 1; for the lactate to pyruvate ratios of 10 and 100, stimulation caused by glucagon was much smaller than that found when Ca2+ was present. e) The effects of glucagon on oxygen uptake in the presence of Ca2+ showed a parabolic relationship with the lactate to pyruvate ratios which was closely similar to that found in the case of gluconeogenesis; the only difference was that inhibition rather than stimulation of oxygen uptake was observed for a lactate to pyruvate ratio equal to 0.01; progressive stimulation was observed in the 0.1 to 100 range. f) In the absence of Ca2+ the effects of glucagon on oxygen uptake were different; the dependence curve was sigmoidal at the onset, with a well-defined maximum at a lactate to pyruvate ratio equal to 1; this maximum was followed by a steady decline at higher ratios; at the ratios of 0.01 and 0.1 inhibition took place; oxygen uptake stimulation caused by glucagon was generally lower in the absence of Ca2+ except when the lactate to pyruvate ratio was equal to 1. The results of the present study demonstrate that stimulation of gluconeogenesis by glucagon depends on Ca2+. However, Ca2+ is only effective in helping gluconeogenesis stimulation by glucagon at highly negative redox potentials of the cytosolic NAD+-NADH system. The triple interdependence of glucagon-Ca2+-NAD+-NADH redox potential reveals highly complex interrelations that can only be partially understood at the present stage of knowledge

Cell lineage relationship in the stomach of normal and genetically manipulated mice

The oxyntic mucosa of the mouse stomach is lined with a heterogeneous population of cells that form numerous short pits continuous with long tubular glands. Tritiated thymidine radioautography has made it possible to pinpoint the origin of all cell types and to follow the differentiation/migration of different cell lineages along the pit-gland unit. The proliferating multipotent stem cells functionally anchored in the upper glandular region, the isthmus, give rise to three main lineage precursors: 1) pre-pit cells, which migrate upward to the pit while differentiating into mucus-producing pit cells; 2) pre-neck cells, which migrate downward to the glandular neck while differentiating into mucus-producing neck cells that, by approaching the glandular base, gradually change their phenotype into pepsinogen- and intrinsic factor-producing zymogenic cells; 3) pre-parietal cells, which differentiate into acid-producing parietal cells in the isthmus and then undergo bipolar migration towards the pit and the glandular base. Thus, parietal cells are the only cells that complete their differentiation in the isthmus and then migrate to be scattered throughout the pit-gland unit. To determine whether parietal cells play a role in controlling decisions about cell fate within the pit-gland unit, the gastric epithelium has been examined in transgenic mice expressing the H,K-ATPase ß-subunit-1035 to +24/simian virus 40 large T antigen fusion gene. The blockade in parietal cell differentiation in these mice produces an amplification of lineage precursors, a marked depletion of zymogenic cells and an increase in pit cell census. Ablation of parietal cells in another transgenic mouse model expressing the H,K-ATPase ß-subunit-1035 to +24/diphtheria toxin fragment A fusion gene also produces amplification of lineage precursors, and similar effects on zymogenic and pit cell census. These findings strongly suggest that parietal cells produce regulatory signals that control the cellular differentiation program of both pit and zymogenic cell lineages, and would hopefully improve our ability to identify the cellular pathways leading to malignant transformation

Cytokines produced by susceptible and resistant mice in the course of Paracoccidioides brasiliensis infection

Paracoccidioidomycosis (PCM) is the most prevalent deep mycosis in Latin America and presents a wide spectrum of clinical manifestations. We established a genetically controlled murine model of PCM, where A/Sn mice develop an infection which mimics the benign disease (immune responses which favor cellular immunity) and B10.A animals present the progressive disseminated form of PCM (preferential activation of B cells and impairment of cellular immune responses). To understand the immunoregulatory phenomena associated with resistance and susceptibility in experimental PCM, A/Sn and B10.A mice were studied regarding antigen-elicited secretion of monokines (TNF-a and TGF-ß) and type-1 (IL-2 and IFN-g) and type-2 (IL-4,5,10) cytokines. Total lymph node cells from resistant mice infected ip with P. brasiliensis produced early and sustained levels of IFN-g and IL-2; type-2 cytokines (IL-4 and IL-5) started to appear 8 weeks after infection. In contrast, susceptible mice produced low levels of IFN-g concomitant with significant levels of IL-5 and IL-10 early in the infection. In the chronic phase of the disease, susceptible animals presented a transitory secretion of IL-2, and IL-4. In the pulmonary infection IL-4, IL-5 and IL-10 were preferentially detected in the lung cells washings of susceptible animals. After in vitro challenge with fungal antigens, normal peritoneal macrophages from B10.A mice secreted high levels of TGF-ß and low levels of TNF-a. In contrast, macrophages from A/Sn animals released high levels of TNF-a associated with a small production of TGF-ß. The in vivo depletion of IFN-g not only abrogated the resistance of A/Sn mice but also diminished the relative resistance of B10.A animals. The in vivo depletion of IL-4 did not alter the disease outcome, whereas administration of rIL-12 significantly enhanced resistance in susceptible animals. Taken together, these results suggest that an early secretion of high levels of TNF-a and IFN-g followed by a sustained secretion of IL-2 and IFN-g plays a dominant role in the resistance mechanisms to P. brasiliensis infection. In contrast, an early and ephemeral secretion of low levels of TNF-a and IFN-g associated with production of IL-5, IL-10 and TGF-ß characterizes the progressive disease of susceptible animals.

The role of natural killer cells in the early period of infection in murine cutaneous leishmaniasis

In order to study the role of natural killer (NK) cells during the early period of Leishmania infection, BALB/c mice were selectively and permanently depleted of NK cells by injection with 90Sr and subsequently infected with Leishmania (Leishmania) amazonensis (HSJD-1 strain). 90Sr is known to selectively deplete NK cells, leaving an intact T- and B-cell compartment and preserving the ability to produce both interferon alpha and IL-2. This method of depletion has advantages when compared with depletion using anti-NK cell monoclonal antibodies because the effect is permanent and neither activates complement nor provokes massive cell death. In the present study, after one month of treatment with 90Sr, the depletion of NK cells was shown by a more than ten-fold reduction in the cytotoxic activity of these cells: 2 x 106 spleen cells from NK-depleted animals were required to reach the same specific lysis of target cells effected by 0.15 x 106 spleen cells from normal control animals. The histopathology of the skin lesion at 7 days after Leishmania infection showed more parasites in the NK cell-depleted group. This observation further strengthens a direct role of NK cells during the early period of Leishmania infection.

DL-methionine supplementation of rice-and-bean diets affects gamma-glutamyltranspeptidase activity and glutathione content in livers of growing rats

Gamma-glutamyltranspeptidase (GGT-EC 2.3.2.2) activity and glutathione (GSH) content were measured in livers of female weanling Wistar rats (N = 5-18), submitted to rice-and-bean diets (13 and 6% w/w protein), both supplemented or not with DL-methionine (0.5 and 0.23 g/100 g dry diet, respectively). After 28 days, the rats on the rice-and-bean diets showed significantly higher levels (four times higher) of liver GGT activity and a concomitant 50% lower concentration of liver GSH in comparison with control groups feeding on casein. The addition of DL-methionine to rice-and-bean diets significantly increased the liver GSH content, which reached levels 50% higher than those found in animals on casein diets. The increase in GSH was accompanied by a decrease in liver GGT activity, which did not reach levels as low as those observed in the control groups. No significant correlation could be established between GGT and GSH changes under the present experimental conditions. Linear correlation analysis only revealed that in animals submitted to unsupplemented rice-and-bean diets GSH concentration was positively associated (P<0.05) with weight gain, food intake and food efficiency. GGT, however, was negatively correlated (P<0.05) with food intake only, and exclusively for supplemented rice-and-bean diets. The high levels of GGT activity observed in the present study for rats receiving a rice-and-bean mixture could be a result of the poor quality of these diets associated with their deficiency in sulfur amino acids. The results also suggest that diet supplementation with methionine could be important in the reduction of the deleterious effects of GSH depletion by restoring the intracellular concentration of this tripeptide.

Detection of specific IgE antibodies in parasite diseases

Activation of Th1 or Th2 cells is associated with production of specific immunoglobulin isotypes, offering the opportunity to use antibody measurement for evaluation of T cell function. Schistosomiasis and visceral leishmaniasis are diseases associated with Th2 activation. However, an IgE response is not always detected in these patients. In the present study we evaluated specific IgE antibodies to S. mansoni and L. chagasi antigens by ELISA after depletion of serum IgG with protein G immobilized on Sepharose beads or RF-absorbent (purified sheep IgG antibodies anti-human IgG). In schistosomiasis patients, specific IgE to SWAP antigen was demonstrable in only 10 of 21 patients (48%) (mean absorbance ± SD = 0.102 ± 0.195) when unabsorbed serum was used. Depletion of IgG with protein G increased the number of specific IgE-positive tests to 13 (62%) and the use of RF-absorbent increased the number of positive results to 20 (95%) (mean absorbances ± SD = 0.303 ± 0.455 and 0.374 ± 0.477, respectively). Specific IgE anti-L. chagasi antibodies were not detected in unabsorbed serum from visceral leishmaniasis patients. When IgG was depleted with protein G, IgE antibodies were detected in only 3 (11%) of 27 patients, and the use of RF-absorbent permitted the detection of this isotype in all 27 visceral leishmaniasis sera tested (mean absorbance ± SD = 0.104 ± 0.03). These data show that the presence of IgG antibodies may prevent the detection of a specific IgE response in these parasite diseases. RF-absorbent, a reagent that blocks IgG-binding sites and also removes rheumatoid factor, was more efficient than protein G for the demonstration of specific IgE antibodies.

Ghost protein damage by peroxynitrite and its protection by melatonin

We have studied the effect of peroxynitrite (ONOO-) on the membrane cytoskeleton of red blood cells and its protection by melatonin. Analysis of the protein fraction of the preparation by SDS-PAGE revealed a dose-dependent (0-600 µM ONOO-) disappearance at pH 7.4 of the main proteins: spectrin, band 3, and actin, with the concomitant formation of high-molecular weight aggregates resistant to reduction by ß-mercaptoethanol (2%) at room temperature for 20 min. These aggregates were not solubilized by 8 M urea. Incubation of the membrane cytoskeleton with ONOO- was characterized by a marked depletion of free sulfhydryl groups (50% at 250 µM ONOO-). However, a lack of effect of ß-mercaptoethanol suggests that, under our conditions, aggregate formation is not mediated only by sulfhydryl oxidation. The lack of a protective effect of the metal chelator diethylenetriaminepentaacetic acid confirmed that ONOO--induced oxidative damage does not occur only by a transition metal-dependent mechanism. However, we demonstrated a strong protection against cytoskeletal alterations by desferrioxamine, which has been described as a direct scavenger of the protonated form of peroxynitrite. Desferrioxamine (0.5 mM) also inhibited the loss of tryptophan fluorescence observed when the ghosts were treated with ONOO-. Glutathione, cysteine, and Trolox® (1 mM), but not mannitol (100 mM), were able to protect the proteins against the effect of ONOO- in a dose-dependent manner. Melatonin (0-1 mM) was especially efficient in reducing the loss of spectrin proteins when treated with ONOO- (90% at 500 µM melatonin). Our findings show that the cytoskeleton, and in particular spectrin, is a sensitive target for ONOO-. Specific antioxidants can protect against such alterations, which could seriously impair cell dynamics and generate morphological changes.

Plasma metanephrines: a novel and cost-effective test for pheochromocytoma

Pheochromocytomas are rare chromaffin cell tumors that nevertheless must be excluded in large numbers of patients who develop sustained or episodic hypertension as well as in many others with suggestive symptoms or with a familial history of pheochromocytoma. Diagnosis of pheochromocytoma depends importantly on biochemical evidence of excess catecholamine production by a tumor. Imperfect sensitivity and specificity of commonly available biochemical tests and the low incidence of the tumor among the tested population mean that considerable time and effort can be expended in confirming or ruling out pheochromocytoma in patients where the tumor is suspected. Measurements of plasma free metanephrines provide a superior test compared to other available tests for diagnosis of pheochromocytoma. In particular, the high sensitivity of plasma free metanephrines means that a normal test result reliably excludes all but the smallest of pheochromocytomas so that no other tests are necessary. Measurements of plasma free metanephrines, when systematically combined with other diagnostic procedures outlined in this review, provide a more efficient, reliable and cost-effective approach for diagnosis of pheochromocytoma than offered by previously available approaches.