Biochemical responses of the ethylene-insensitive Never ripe tomato mutant subjected to cadmium and sodium stresses

In order to further address the known interaction between ethylene and components of the oxidative system, we have used the ethylene-insensitive Never ripe (Nr) tomato (Solanum lycopersicum L) mutant, which blocks ethylene responses. The mutant was compared to the control Micro-Tom (MT) cultivar subjected to two stressful situations: 100 mM NaCl and 0.5 mM CdCl(2). Leaf chlorophyll, lipid peroxidation and antioxidant enzyme activities in roots, leaves and fruits, and Na and Cd accumulation in tissues were determined. Although we verified a similar growth pattern and Na and Cd accumulation for MT and Nr, the mutant exhibited reduced leaf chlorophyll degradation following stress. In roots and leaves, the patterns of catalase (CAT), glutathione reductase (GR), ascorbate peroxidase (APX), guaiacol peroxidase (GPOX), superoxide dismutase (SOD) enzyme activity as well as malondialdehyde (MDA) and hydrogen peroxide (H(2)O(2)) production under the stressful conditions tested were very similar between MT and Nr mutant. However, Nr fruits showed increased H(2)O(2) production, reduced and enhanced APX activity in NaCl and CdCl(2), respectively, and enhanced GPOX in NaCl. Moreover, through non-denaturing PAGE, a similar reduction of SOD I band intensity in both, control MT and Nr mutant, treated with NaCl was observed. In leaves and fruits, a similar SOD activity pattern was observed for all periods, genotypes and treatments. Overall the results indicate that the ethylene signaling associated with NR receptor can modulate the biochemical pathways of oxidative stress in a tissue dependent manner, and that this signaling may be different following Na and Cd exposure. (C) 2011 Elsevier B.V. All rights reserved.

Effects of the herbicides acetochlor and metolachlor on antioxidant enzymes in soil bacteria

The aim of this study was to investigate the antioxidant responses of three bacteria (SD1. KD and K9) isolated from soil previously treated with the herbicides metolachlor and acetochlor. By 165 rRNA gene sequencing, we determined that SD1 is phylogenetically related to Enterobacter asburiae, while KD and K9 have divergent genomes that more closely resemble that of Enterobacter amnigenus. Decreased levels of lipid peroxidation were observed in SD1 and KD following treatment with 34 mM metolachlor or 62 mM acetochlor, respectively, indicating that both bacteria were able to adapt to an increase in ROS production. In the presence of 34 mM metolachlor or 62 mM acetochlor, all bacterial isolates exhibited increases in total catalase (CAT) activity (81% for SDI, 53% for KD and 59% for K9), whereas total SOD activity (assessed based on the profile and intensity of the bands) was slightly reduced when the bacteria were exposed to high concentrations of the herbicides (340 mM metolachlor or 620 mM acetochlor). This effect was due to a specific reduction in SOD IV (K9 and KD isolates) by 45% and 90%, respectively, and SOD V (SD1 isolate) isoenzymes by 60%. The most striking result was obtained in the SD1 isolate, where two novel isoenzymes of glutathione reductase (GR) that responded specifically to metolachlor were identified. In addition, acetochlor was shown to induce the expression of a new 57 kDa protein band in the K9 and KD isolates. The bacteria isolated from the herbicide-contaminated soil exhibited an efficient antioxidant system response at herbicide concentrations of up to 34 mM metolachlor or 62 mM acetochlor. These data suggest a mechanism for tolerance that may include the control of an imbalance in ROS production versus scavenging. The data suggest that specific isoenzymes of CAT and GR could be involved in this herbicide tolerance mechanism. (C) 2011 Elsevier Ltd. All rights reserved.

Enantioselective analysis of propranolol and 4-hydroxypropranolol by CE with application to biotransformation studies employing endophytic fungi

A CE method is described for the enantioselective analysis of propranolol (Prop) and 4-hydroxypropranolol (4-OH-Prop) in liquid Czapek medium with application in the study of the enantioselective biotransformation of Prop by endophytic fungi. The electrophoretic conditions previously optimized were as follows: an uncoated fused-silica capillary, 4%w/v carboxymethyl-beta-CD in 25 mmol/L triethylamine/phosphoric acid (H(3)PO(4)) buffer at pH 9 as running electrolyte and 17 kV of voltage. UV detection was carried out at 208 nm. Liquid-liquid extraction using diethyl ether: ethyl acetate (1:1 v/v) as extractor solvent was employed for sample preparation. The calibration curves were linear over the concentration range of 0.25-10.0 mu g/mL for each 4-OH-Prop enantiomer and 0.10-10.0 mu g/mL for each Prop enantiomer (r >= 0.995). Within-day and between-day relative standard deviations and relative errors for precision and accuracy were lower than 15% for all the enantiomers. Finally, the validated method was used to evaluate Prop biotransformation in its mammalian metabolite 4-OH-Prop by some selected endophytic fungi. The screening of five strains of endophytic fungi was performed and all of them could biotransform Prop to some extent. Specifically, Glomerella cingulata (VA1) biotransformed 47.8% of (-)-(S)-Prop to (-)-(S)-4-OH-Prop with no formation of (+)-(R)4-OH-Prop in 72 h of incubation.

Biochemical and Functional Characterization of a Metalloprotease from the Thermophilic Fungus Thermoascus aurantiacus

Protease production was carried out in solid state fermentation. The enzyme was purified through precipitation with ethanol at 72% followed by chromatographies in columns of Sephadex G75 and Sephacryl S100. It was purified 80-fold and exhibited recovery of total activity of 0.4%. SDS-PAGE analysis indicated an estimated molecular mass of 24.5 kDa and the N-terminal sequence of the first 22 residues was APYSGYQCSMQLCLTCALMNCA. Purified protease was only inhibited by EDTA (96.7%) and stimulated by Fe(2+) revealing to be a metalloprotease activated by iron. Optimum pH was 5.5, optimum temperature was 75 degrees C, and it was thermostable at 65 degrees C for 1 h maintaining more than 70% of original activity. Through enzyme kinetic studies, protease better hydrolyzed casein than azocasein. The screening of fluorescence resonance energy transfer (FRET) peptide series derived from Abz-KLXSSKQ-EDDnp revealed that the enzyme exhibited preference for Arg in P(1) (k(cat)/K(m) = 30.1 mM(-1) s(-1)).

Box-Behnken design for the optimization of an enantioselective method for the simultaneous analysis of propranolol and 4-hydroxypropranolol by CE

An experimental design optimization (Box-Behnken design, BBD) was used to develop a CE method for the simultaneous resolution of propranolol (Prop) and 4-hydroxypropranolol enantiomers and acetaminophen (internal standard). The method was optimized using an uncoated fused silica capillary, carboxymethyl-beta-cyclodextrin (CM-beta-CD) as chiral selector and triethylamine/phosphoric acid buffer in alkaline conditions. A BBD for four factors was selected to observe the effects of buffer electrolyte concentration, pH, CM-beta-CD concentration and voltage on separation responses. Each factor was studied at three levels: high, central and low, and three center points were added. The buffer electrolyte concentration ranged from 25 to 75 mM, the pH ranged from 8 to 9, the CM-beta-CD concentration ranged from 3.5 to 4.5%w/v, and the applied run voltage ranged from 14 to 20 W. The responses evaluated were resolution and migration time for the last peak. The obtained responses were processed by Minitab (R) to evaluate the significance of the effects and to find the optimum analysis conditions. The best results were obtained using 4%w/v CM-beta-CD in 25 mM triethylamine/H(3)PO(4) buffer at pH 9 as running electrolyte and 17 kV of voltage. Resolution values of 1.98 and 1.95 were obtained for Prop and 4-hydroxypropranolol enantiomers, respectively. The total analysis time was around of 15 min. The BBD showed to be an adequate design for the development of a CE method, resulting in a rapid and efficient optimization of the pH and concentration of the buffer, cyclodextrin concentration and applied voltage.

An evaluation, using the comet assay and the micronucleus test, of the antigenotoxic effects of chlorophyll b in mice

We investigated the effects of the dietary pigment chlorophyll b (CLb) on cisplatin (cDDP)-induced oxidative stress and DNA damage, using the comet assay in mouse peripheral blood cells and the micronucleus (MN) test in bone marrow and peripheral blood cells. We also tested for thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH) in liver and kidney tissues, as well as catalase (CAT) activity and GSH in total blood. CLb (0.2 and 0.5 mg/kg b.w.) was administrated by gavage every day for 13 days. On the 14th day of the experiment, 6 mg/kg cDDP or saline was delivered intraperitoneally. Treatment with cDDP led to a significant decrease in DNA migration and an increase in MN frequency in both cell types, bone marrow and peripheral blood cells. In the kidneys of mice treated with cDDP, TBARS levels were increased, whereas GSH levels were depleted in kidney and liver. In mice that were pretreated with CLb and then treated with cDDP, TBARS levels maintained normal concentrations and GSH did not differ from cDDP group. The improvement of oxidative stress biomarkers after CLb pre-treatment was associated with a decrease in DNA damage, mainly for the highest dose evaluated. Furthermore, CLb also slightly reduced the frequency of chromosomal breakage and micronucleus formation in mouse bone marrow and peripheral blood cells. These results show that pre-treatment with CLb attenuates cDDP-induced oxidative stress, chromosome instability, and lipid peroxidation. (C) 2011 Elsevier B.V. All rights reserved.

Low level and sub-chronic exposure to methylmercury induces hypertension in rats: nitric oxide depletion and oxidative damage as possible mechanisms

Increased risk of hypertension after methylmercury (MeHg) exposure has been suggested. However, the underlying mechanisms are not well explored. In this paper, we have analyzed whether sub-chronic exposure to MeHg increases systolic blood pressure even at very low levels. In addition, we analyzed if the methylmercury-induced hypertension is associated with a decreased plasmatic nitric oxide levels and with a dysregulation of the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), as well as the levels of MDA and glutathione. For this study, Wistar rats were treated with methylmercury chloride (100 mu g/kg per day) or vehicle. Total treatment time was 100 days. Malondialdehyde (MDA) and circulating NOx levels and superoxide dismutase (SOD) and catalase (CAT) activities were determined in plasma, whereas glutathione levels were determined in erythrocytes. Our results show that long-term treatment at a low level of MeHg affected systolic blood pressure, increasing and reducing the levels of plasmatic MDA and NOx, respectively. However, the activity of SOD did not decrease in the MeHg exposed group when compared to the control. We found a negative correlation between plasmatic nitrite/nitrate (NOx) levels and systolic blood pressure (r = -0.67; P = 0.001), and a positive correlation between MDA and systolic blood pressure (r = 0.61; P = 0.03), thus suggesting increased inhibition of NO formation with the increase of hypertension. In conclusion, long-term exposure to a low dose of MeHg increases the systolic pressure and is associated, at least in part, with increased production of ROS as judged by increased production of malondialdehyde and depressed NO availability.

Protective properties of quercetin against DNA damage and oxidative stress induced by methylmercury in rats

Aim of the study was to find out whether consumption of quercetin (QC), an abundant flavonoid in the human diet, protects against DNA damage caused by exposure to organic mercury. Therefore, rats were treated orally with methylmercury (MeHg) and the flavonoid with doses that reflect the human exposure. The animals received MeHg (30 mu g/kg/bw/day), QC (0.5-50 mg/kg/bw/day), or combinations of both over 45 days. Subsequently, the glutathione levels (GSH) and the activities of glutathione peroxidase (GPx) and catalase (CAT) were determined, and DNA damage was measured in hepatocytes and peripheral leukocytes in single cell gel electrophoresis assays. MeHg decreased the concentration of GSH and the activity of GPx by 17 and 12%, respectively and caused DNA damage to liver and blood cells, while with QC no such effects were seen. When the flavonoid was given in combination with MeHg, the intermediate and the highest concentrations (5.0 and 50.0 mg/kg/bw/day) were found to cause DNA protection; DNA migration was reduced by 54 and 65% in the hepatocytes and by 27 and 36% in the leukocytes; furthermore, the reduction in GSH and GPx levels caused by MeHg treatment was restored. In summary, our results indicate that consumption of QC-rich foods may protect Hg-exposed humans against the adverse health effects of the metal.

Lutein improves antioxidant defense in vivo and protects against DNA damage and chromosome instability induced by cisplatin

Lutein (LT) is the second most prevalent carotenoid in human serum, and it is abundantly present in dark, leafy green vegetables. The objectives of this study were to evaluate the genotoxicity and mutagenicity of LT, and its protective effects in vivo against DNA damage and chromosome instability induced by cisplatin (cDDP). For this purpose, we used the comet assay and micronucleus (MN) test, and we evaluated the antioxidant effects of LT by determination of enzymatic (catalase-CAT) and non-enzymatic (reduced glutathione-GSH) activity. Mice were divided into six groups: cDDP, mineral oil (OM), LT groups and LT + cDDP groups. To perform the MN test on peripheral blood (PB) cells, blood samples were collected before the first treatment (T0), and 36 h (T1) and 14 days (T2) after the first treatment. To perform the comet assay, blood samples were collected 4 h after the first and the last treatment. Oxidative capacity was analyzed in total blood that was collected 24 h after the last treatment, when bone marrow (BM) sample was also collected for the MN test. No genotoxic or mutagenic effects of LT were observed for the doses evaluated. We did find that this carotenoid was able to reduce the formation of crosslinks and chromosome instability induced by cDDP. No differences were observed in CAT levels, and LT treatment increased GSH levels compared with a negative control group, reinforcing the role of this carotenoid as an antioxidant.

The use of complementary and alternative therapies by patients with cancer

The aim of this research was to assess the prevalence and predictors of complementary and alternative therapy (CAT) use among cancer patients in Australia. A total of 1492 cancer patients attending nine major public cancer treatment centers in New South Wales, Australia, were asked to complete the Supportive Care Needs Survey. Of the 1354 consenting patients, 888 (65%) returned a completed survey. This article reports the secondary analyses of the survey data, specifically focusing on CAT use. For all cancers, 17.1% of patients were using at least one CAT. The two main demographic characteristics of CAT users were gender and age, where females were more likely to use CAT than males and that CAT use declined as age increased. Time since diagnosis was identified as the only significant clinical predictor of CAT use, where CAT use increased with time until 5 years since diagnosis. Our research shows that herbal treatments and naturopathy are the most popular CAT used by cancer patients (constituting over 30% of all CAT use recorded). The use of CAT among cancer patients is a significant issue in cancer care, especially considering the potential interactions between CAT and conventional medicines. Given that many cancer patients may not be aware of potential risks associated with these interactions it is important that oncologists and others involved in cancer patient care are informed about CAT and its use amongst their patients.

Targeting of a cholecystokinin-DNA complex to pancreatic cells in vitro and in vivo

The carboxy terminal octapeptide of cholecystokinin (CCK8) is a hormone that binds high affinity receptors in a number of tissues including pancreas and pancreatic tumours. As part of our studies to develop effective gene therapy for the treatment of pancreatic cancers, we have investigated various gene delivery systems that depend on CCK8 receptor targeting. In this paper,we describe the synthesis of a CCK8-DNA complex designed to deliver foreign DNA to cholecystokinin receptor-positive cells. CCK8 was ligated to avidin and then complexed to linearis biotinylated DNA (pSV-CAT). The uptake of P-32-labelled CCK8-DNA complex by rat pancreatic acini was linear with time over 4 h with 65-70% of uptake inhibited by 100 nM CCK8. The complex appeared to be internalised since it could not be removed by acid wash. When administered intra-arterially, the complex was rapidly removed from the circulation with no evidence of targeted delivery to the pancreas, However, following a single intraperitoneal dose, the pancreas accumulated-5- 8% of the total administered complex by 24 h. These results suggest that peptide-dependent gene delivery to CCK receptor positive cells in vivo is feasible but, when administered directly into the circulation, diffusional barriers across the endothelium may limit distribution to peripheral tissues. Intraperitoneal administration therefore may be a useful alternative for targeting the pancreas.

Cat-scratch disease: A case report

Cat-scratch disease is the most common cause of benign lymphadenopathy in children and young adults. A 21-year-old patient presented with a 14-day history of asymptomatic left submental swelling. Clinical and radiographic examination revealed no dental cause. An enlarged, firm nodule was excised. The histopathologic appearance was consistent with cat-scratch disease. This article summarizes the relevant literature and discusses diagnosis and treatment of cat-scratch disease.

Accuracy of predicting a child's response to potential threat: A comparison of children and their mothers

Considerable research has indicated that children and their parents often demonstrate marked discrepancies in their reporting of anxiety-related phenomena. In such cases, the question arises as to whether children are capable of accurately reporting on their anxiety. In the present study, 50 children (aged 5 to 14 years) were asked to approach a large, German Shepherd dog. Prior to the task, both the mother and child independently predicted the closest point likely to be reached by the child and the degree of anxiety likely to be experienced. These predictions were then compared with the actual phenomena displayed by the child during the task. On the behavioural measure (closest step reached), both the child and mother demonstrated equivalent predictive accuracy. On the subjective measure (fear ratings) children were considerably more accurate than their mothers. The data were not influenced by gender, age, or clinical status. The results indicate the ability of children to accurately predict their anxious responses, and support the value of incorporating children's self-reports in the assessment of emotional disorders.

Effects of an oral vasopressin receptor antagonist (OPC-31260) in a dog with syndrome of inappropriate secretion of antidiuretic hormone

Objective The syndrome of inappropriate secretion of antidiuretic hormone is a rare disorder in dogs characterised by hypo-osmolality and persistent arginine vasopressin production in the absence of hypovolaemia and/or hypotension. The study describes the efficacy and safety of the nonpeptide selective arginine vasopressin V-2 receptor antagonist OPC-31260 in a dog with the naturally occurring syndrome. Design The detailed case history of a dog with spontaneous syndrome of inappropriate secretion of antidiuretic hormone that received long-term therapy with oral OPC-31260 is presented. Effects of the first dose of OPC-31260 and of a dose administered after a continuous dosing period of 12 days are reported. Procedure Packed cell volume, plasma sodium, total protein, arginine vasopressin, renin activity, atrial natriuretic peptide, urine specific gravity, urine output, heart rate and body weight were monitored for 2 h before, and for 4 h after, the first dose of OPC-31260. The same parameters plus plasma osmolality and urine osmolality were monitored when an identical dose was administered after 12 days of therapy. Results Oral administration of OPC-31260 at 3 mg/kg body weight resulted in marked aquaresis with increased urine output and decline in urine specific gravity within 1 h. Corresponding increases in concentrations of plasma sodium, plasma osmolality and plasma renin activity were recorded over a 4 h period. Arginine vasopressin concentration remained inappropriately elevated throughout the study. Results were similar when the trial procedure was repeated after a stabilisation period of 12 days. Long-term therapy with OPC-31260 at a dose of 3 mg/kg body weight orally every 12 h resulted in good control of clinical signs with no deleterious effects detected during a 3-year follow-up period. Despite sustained clinical benefits observed in this case, plasma sodium did not normalise with continued administration of the drug. Conclusions Treatment of a dog with naturally occurring syndrome of inappropriate secretion of antidiuretic hormone with OPC-31260 at 3 mg/kg body weight orally every 12 h resulted in marked aquaresis and significant palliation of clinical signs with no discernible side-effects detected over a 3-year period. Thus, OPC-31260 appears to offer a feasible medical alternative to water restriction for treatment of dogs with syndrome of inappropriate secretion of antidiuretic hormone. Higher doses of OPC-31260 may be required to achieve and maintain normal plasma sodium in dogs with this syndrome.

H-reflex modulation during passive lengthening and shortening of the human triceps surae

1. The present study investigated the effects of lengthening and shortening actions on IT-reflex amplitude. H-reflexes were evoked in the soleus (SOL) and medial gastroenemius (MG) of human subject, during passive isometric, lengthening and shortening actions performed at angular velocities of 0, +/-2, +/-5 and +/- 15 deg s(-1). 2. H-reflex amplitude, in froth SOL and MG were significantly depressed during passive lengthening actions and facilitated during passive shortening actions, when compared with the isometric R-reflex amplitude. 3. Four experiments were performed in which the latencies front the onset of movement to delivery of the stimulus were altered. Passive H-reflex modulation during lengthening actions was found tee begin at latencies of less than 60 ms suggesting that this inhibition was due to peripheral and/or spinal mechanisms. 4. It is postulated that, the H-reflex modulation seen in the present study is related to the tunic discharge of muscle spindle afferents and the consequent effects of transmission within the la pathway. Inhibition of the H-reflex at less than 60 ms after the onset of muscle lengthening may he attributed to several mechanisms, which cannot be distinguished using the current protocol. These may include the inability to evoke volleys in la fibres that are refractory following muscle spindle discharge during; rapid muscle lengthening, a reduced probability of transmitter release front the presynaptic terminal (homosynaptic post.-activation depression) and presynaptic inhibition of la afferents from plantar flexor agonists. Short latency facilitation of the H-reflex may be attributed to temporal summation of excitatory postsynaptic potentials arising from muscle spindle afferents during rapid muscle lengthening. At longer latencies, presynaptic inhibition of Ia afferents cannot be excluded as a potential inhibitory mechanism.