Competitiveness of ALS inhibitors resistant and susceptible biotypes of Greater Beggarticks (Bidens subalternans)

The continuous use of ALS-inhibiting herbicides has led to the evolution of herbicide-resistant weeds worldwide. Greater beggarticks is one of the most troublesome weeds found in the soybean production system in Brazil. Recently, a greater beggarticks biotype that is resistant (R) to ALS inhibitors due to Trp574Leu mutation in the ALS gene was identified. Also, the adaptive traits between susceptible (S) and R to ALS inhibitors biotypes of greater beggarticks were compared. Specifically, we aimed to: (1) evaluate and compare the relative growth rates (RGR) between the biotypes; (2) analyze the seed germination characteristics of R and S biotypes under different temperature conditions; and (3) evaluate their competitive ability in a replacement series study. The experiments were conducted at the University of Arkansas, USA, in 2007 and at Universidade Federal do Rio Grande do Sul (Federal University of Rio Grande do Sul), Brazil, in 2008. Plant proportions for replacement series studies were respectively 100:0, 75:25, 50:50, 25:75 and 0:100, with a total population of 150 plants m-2. There was no difference in RGR between R and S biotypes. The R-biotype germination rate was lower than that of the S biotype. However, at low temperature conditions (15 ºC), the reverse was observed. In general, there is no difference in the competitive ability between R and S greater beggarticks biotypes.

Eletrolite leakage as a technique to diagnose euphorbia heterophylla biotypes resistant to ppo-inhibitors herbicides

The action of herbicides that affect the integrity of cell membranes and cause leakage, like PPO-inhibitors, can be detected by measuring the electric conductivity (EC) of a solution in which the plant tissue target is incubated in the presence of herbicide. The objectives of this work were to confirm PPO resistance in a new Euphorbia heterophylla (EPHHL) biotype, and to compare the electrolyte leakage from R and S to PPO-inhibitors biotypes, using two different methods of incubation in a solution containing herbicides. One experiment was carried in greenhouse and three in laboratory, with a completely randomized design. In the greenhouse experiment, four biotypes of EPHHL were sprayed with seven rates of fomesafen to confirm resistance in suspected biotypes. Leaf disks from R and S EPHHL biotypes in the second and the third experiments and entire leaves in the fourth experiment were incubated in a solution containing PPO-inhibitors to subsequently determine EC of solution. The study confirmed the resistance to PPO-inhibitors in two EPHHL biotypes. There were no significant differences between S and R biotypes in the experiments with the incubation of leaf disks, but incubation of entire leaves of EPHHL S biotype showed higher EC when in a solution with fomesafen, in comparison to the R biotype. The results of this work are an indirect evidence that resistance to PPO-inhibitors is related to lower absorption of herbicide by the shoots and also to some kind of mechanism to cope with oxidative stress.

Resistance to ACCase inhibitors in Eleusine indica from Brazil involves a target site mutation

Eleusine indica (goosegrass) is a diploid grass weed which has developed resistance to ACCase inhibitors during the last ten years due to the intensive and frequent use of sethoxydim to control grass weeds in soybean crops in Brazil. Plant dose-response assays confirmed the resistant behaviour of one biotype obtaining high resistance factor values: 143 (fenoxaprop), 126 (haloxyfop), 84 (sethoxydim) to 58 (fluazifop). ACCase in vitro assays indicated a target site resistance as the main cause of reduced susceptibility to ACCase inhibitors. PCR-generated fragments of the ACCase CT domain of the resistant and sensitive reference biotype were sequenced and compared. A point mutation was detected within the triplet of aspartate at the amino acid position 2078 (referred to EMBL accession no. AJ310767) and resulted in the triplet of glycine. These results constitute the first report on a target site mutation for a Brazilian herbicide resistant grass weed.

Growth inhibitors in turfgrass

Well-maintained lawns are comfortable and safe places for leisure activities and sports practice, and they also bring environmental benefits; for example, they reduce soil exposure to erosion and releases atmospheric CO2, thus reducing the greenhouse effect. However, regardless of the purpose of use or the choice of the plant species to form the lawn, the highest costs involve cutting that is needed to keep the turfgrass at its appropriate height. Successive lawn cutting operations are necessary basically because of the vegetative and reproductive growth of turfgrass which, in Brazil, occurs mainly from October to March. Expenditures with successive mechanical cuttings have fostered the search of alternative procedures to keep lawn plants at appropriate height, such as the use of plant growth inhibitors, an increasingly interesting procedure. Since the use of this technology in Brazil is still at its early stage, the aim of this literature review is to examine aspects associated with lawn management by using growth inhibitors. Another alternative is to increase the knowledge of the classification and rational application of the different compounds currently available in the market.

The study of genetic polymorphisms related to serotonin in Alzheimer's disease: a new perspective in a heterogenic disorder

Genetic and environmental factors have been implicated in the development of Alzheimer's disease (AD), the most common form of dementia in the elderly. Mutations in 3 genes mapped on chromosomes 21, 14 and 1 are related to the rare early onset forms of AD while the e4 allele of the apolipoprotein E (APOE) gene (on chromosome 19) is the major susceptibility locus for the most common late onset AD (LOAD). Serotonin (5-hydroxytryptamine or 5-HT) is a key neurotransmitter implicated in the control of mood, sleep, appetite and a variety of traits and behaviors. Recently, a polymorphism in the transcriptional control region upstream of the 5-HT transporter (5-HTT) gene has been studied in several psychiatric diseases and personality traits. It has been demonstrated that the short variant(s) of this 5-HTT gene-linked polymorphic region (5-HTTLPR) is associated with a different transcriptional efficiency of the 5-HTT gene promoter resulting in decreased 5-HTT expression and 5-HT uptake in lymphocytes. An increased frequency of this 5-HTTLPR short variant polymorphism in LOAD was recently reported. In addition, another common polymorphic variation in the 5-HT2A and 5-HT2C serotonin receptor genes previously analyzed in schizophrenic patients was associated with auditory and visual hallucinations in AD. These observations suggest that the involvement of the serotonin pathway might provide an explanation for some aspects of the affective symptoms commonly observed in AD patients. In summary, research on genetic polymorphisms related to AD and involved in receptors, transporter proteins and the enzymatic machinery of serotonin might enhance our understanding of this devastating neurodegenerative disorder.

Effects of serotonin and fluoxetine on blood glucose regulation in two decapod species

One of the best known crustacean hormones is the crustacean hyperglycemic hormone (CHH). However, the mechanisms involved in hormone release in these animals are poorly understood, and thus constitute the central objective of the present study. Different groups of crustaceans belonging to diverse taxa (Chasmagnathus granulata, a grapsid crab and Orconectes limosus, an astacid) were injected with serotonin, fluoxetine, or a mixture of both, and glycemic values (C. granulata and O. limosus) and CHH levels (O. limosus) were determined after 2 h in either submerged animals or animals exposed to atmospheric air. Both serotonin and fluoxetine caused significant hyperglycemia (P<0.05) after injection into the blood sinus of the two species, an effect enhanced after exposure to atmospheric air. In C. granulata blood glucose increased from 6.1 to 43.3 and 11.4 mg/100 ml in submerged animals and from 5.7 to 55.2 and 22.5 mg/100 ml in air-exposed animals after treatment with serotonin and fluoxetine, respectively. In O. limosus the increases were from 1.2 to 59.7 and 135.2 mg/100 ml in submerged animals and from 2.5 to 200.3 and 193.6 mg/100 ml in air-exposed animals after treatment with serotonin and fluoxetine, respectively. Serotonin and fluoxetine also caused a significant increase in the circulating levels of CHH in O. limosus, from 11.9 to 43 and 45.7 fmol/ml in submerged animals and from 13.2 to 32.6 and 45.7 fmol/ml in air-exposed animals, respectively, thus confirming their action as neuroregulators in these invertebrates.

Sex hormone modulation of serotonin-induced coronary vasodilation in isolated heart

The present study was designed to evaluate the differences in the coronary vasodilator actions of serotonin (5-HT) in isolated heart obtained from naive or castrated male and female rats that were treated with either estrogen or testosterone. Hearts from 12 groups of rats were used: male and female naive animals, castrated, castrated and treated with 17ß-estradiol (0.5 µg kg-1 day-1) for 7 or 30 days, and castrated and treated with testosterone (0.5 mg kg-1 day-1) for 7 or 30 days. After treatment, the vascular reactivity of the coronary bed was evaluated. Baseline coronary perfusion pressure (CPP) was determined and dose-response curves to 5-HT were generated. Baseline CPP differed between male (70 ± 6 mmHg, N = 10) and female (115 ± 6 mmHg, N = 12) naive rats. Maximal 5-HT-induced coronary vasodilation was higher (P<0.05) in naive female than in naive male rats. In both sexes, 5-HT produced endothelium-dependent coronary vasodilation. After castration, there was no significant difference in baseline CPP between hearts obtained from male and female rats (75 ± 7 mmHg, N = 8, and 83 ± 5 mmHg, N = 8, respectively). Castration reduced the 5-HT-induced maximal vasodilation in female and male rats (P<0.05). Estrogen treatment of castrated female rats restored (P<0.05) the vascular reactivity. In castrated male rats, 30 days of estrogen treatment increased (P<0.05) the responsiveness to 5-HT. The endothelium-dependent coronary vasodilator actions of 5-HT are greater in female rats and are modulated by estrogen. A knowledge of the mechanism of action of estrogen on coronary arteries could aid in the development of new therapeutic strategies and potentially decrease the incidence of cardiovascular disease in both sexes.

DNA topoisomerase inhibitors: biflavonoids from Ouratea species

Topoisomerase inhibitors are agents with anticancer activity. 7"-O-Methyl-agathisflavone (I) and amentoflavone (II) are biflavonoids and were isolated from the Brazilian plants Ouratea hexasperma and O. semiserrata, respectively. These biflavonoids and the acetyl derivative of II (IIa) are inhibitors of human DNA topoisomerases I at 200 µM, as demonstrated by the relaxation assay of supercoiled DNA, and only agathisflavone (I) at 200 µM also inhibited DNA topoisomerases II-alpha, as observed by decatenation and relaxation assays. The biflavonoids showed concentration-dependent growth inhibitory activities on Ehrlich carcinoma cells in 45-h culture, assayed by a tetrazolium method, with IC50 = 24 ± 1.4 µM for I, 26 ± 1.1 µM for II and 10 ± 0.7 µM for IIa. These biflavonoids were assayed against human K562 leukemia cells in 45-h culture, but only I showed 42% growth inhibitory activity at 90 µM. Our results suggest that biflavonoids are targets for DNA topoisomerases and their cytotoxicity is dependent on tumor cell type.

A brain microdialysis study on 5-HT release in freely moving rat lines selectively bred for differential 5-HT1A receptor function

Breeding for high and low hypothermic responses to systemic administration of a serotonin1A (5-HT1A) receptor agonist (8-hydroxy-2-(di-n-propylamino)tetralin, 8-OH-DPAT) has resulted in high DPAT-sensitive (HDS) and low DPAT-sensitive (LDS) lines of rats, respectively. These lines also differ in several behavioral measures associated with stress. In the present microdialysis study we observed that basal 5-HT concentrations in the prefrontal cortex and dorsal hippocampus did not differ significantly between HDS and LDS rats. Thus, behavioral differences between the HDS and LDS lines might not be attributed to differences in basal 5-HT release. However, both lines had lower basal levels of 5-HT release than their randomly bred control group (random DPAT-sensitive, RDS) in the prefrontal cortex (mean ± SEM, pg/20 µl, was 3.0 ± 0.4 for LDS, 3.8 ± 0.3 for HDS and 6.4 ± 0.6 for RDS; F(2,59) = 5.8, P<0.005). The administration of (±)-fenfluramine (10 mg/kg) induced a greater increase in hippocampal 5-HT levels in HDS rats (500%) as compared with LDS (248%) or RDS (243%) rats (P<0.0001). There were no significant differences in the prefrontal cortex among lines, with a fenfluramine-induced 5-HT increase of about 900% in the three groups. This differential response to fenfluramine may be due to functional alterations of hippocampal 5-HT reuptake sites in the HDS line.

Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors

Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 µg/ml) on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates) reduced (1.2 to 3.0 times) the catalytic efficiency of kallikrein (in a nanomolar range) on the hydrolysis of plasminogen (0.3 to 1.8 µM) and increased (1.9 to 7.7 times) the enzyme efficiency in factor XII (0.1 to 10 µM) activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times) kallikrein inhibition by antithrombin (1.4 µM), while chondroitin 4- and 6-sulfates reduced it (1.3 times). Heparin and heparan sulfate increased (1.4 times) the enzyme inhibition by the C1-inhibitor (150 nM).

Interruption of the blood-stage cycle of the malaria parasite, Plasmodium chabaudi, by protein tyrosine kinase inhibitors

Malaria is a devastating disease caused by a unicellular protozoan, Plasmodium, which affects 3.7 million people every year. Resistance of the parasite to classical treatments such as chloroquine requires the development of new drugs. To gain insight into the mechanisms that control Plasmodium cell cycle, we have examined the effects of kinase inhibitors on the blood-stage cycle of the rodent malaria parasite, Plasmodium chabaudi. In vitro incubation of red blood cells for 17 h at 37ºC with the inhibitors led to a decrease in the percent of infected cells, compared to control treatment, as follows: genistein (200 µM - 75%), staurosporine (1 µM - 58%), R03 (1 µM - 75%), and tyrphostins B44 (100 µM - 66%) and B46 (100 µM - 68%). All these treatments were shown to retard or prevent maturation of the intraerythrocytic parasites. The diverse concentration ranges at which these inhibitors exert their effects give a clue as to the types of signals that initiate the transitions between the different developmental stages of the parasite. The present data support our hypothesis that the maturation of the intraerythrocytic cycle of malaria parasites requires phosphorylation. In this respect, we have recently reported a high Ca2+ microenvironment surrounding the parasite within red blood cells. Several kinase activities are modulated by Ca2+. The molecular identification of the targets of these kinases could provide new strategies against malaria.

Melatonin and N-acetyl-serotonin cross the red blood cell membrane and evoke calcium mobilization in malarial parasites

The duration of the intraerythrocytic cycle of Plasmodium is a key factor in the pathogenicity of this parasite. The simultaneous attack of the host red blood cells by the parasites depends on the synchronicity of their development. Unraveling the signals at the basis of this synchronicity represents a challenging biological question and may be very important to develop alternative strategies for therapeutic approaches. Recently, we reported that the synchrony of Plasmodium is modulated by melatonin, a host hormone that is synthesized only during the dark phases. Here we report that N-acetyl-serotonin, a melatonin precursor, also releases Ca2+ from isolated P. chabaudi parasites at micro- and nanomolar concentrations and that the release is blocked by 250 mM luzindole, an antagonist of melatonin receptors, and 20 mM U73122, a phospholipase C inhibitor. On the basis of confocal microscopy, we also report the ability of 0.1 µM melatonin and 0.1 µM N-acetyl-serotonin to cross the red blood cell membrane and to mobilize intracellular calcium in parasites previously loaded with the fluorescent calcium indicator Fluo-3 AM. The present data represent a step forward into the understanding of the signal transduction process in the host-parasite relationship by supporting the idea that the host hormone melatonin and N-acetyl-serotonin generate IP3 and therefore mobilize intracellular Ca2+ in Plasmodium inside red blood cells.

Effect of cyclooxygenase inhibitors on gentamicin-induced nephrotoxicity in rats

The frequent use of nonsteroidal anti-inflammatory drugs (NSAID) in combination with gentamicin poses the additional risk of nephrotoxic renal failure. Cyclooxygenase-1 (COX-1) is the main enzyme responsible for the synthesis of renal vasodilator prostaglandins, while COX-2 participates predominantly in the inflammatory process. Both are inhibited by non-selective NSAID such as indomethacin. Selective COX-2 inhibitors such as rofecoxib seem to have fewer renal side effects than non-selective inhibitors. The objective of the present study was to determine whether the combined use of rofecoxib and gentamicin can prevent the increased renal injury caused by gentamicin and indomethacin. Male Wistar rats (250-300 g) were treated with gentamicin (100 mg/kg body weight, ip, N = 7), indomethacin (5 mg/kg, orally, N = 7), rofecoxib (1.4 mg/kg, orally, N = 7), gentamicin + rofecoxib (100 and 1.4 mg/kg, respectively) or gentamicin + indomethacin (100 and 5 mg/kg, respectively, N = 8) for 5 days. Creatinine clearance and alpha-glutathione-S-transferase concentrations were used as markers of renal injury. Animals were anesthetized with ether and sacrificed for blood collection. The use of gentamicin plus indomethacin led to worsened renal function (0.199 ± 0.019 ml/min), as opposed to the absence of a nephrotoxic effect of rofecoxib when gentamicin plus rofexicob was used (0.242 ± 0.011 ml/min). These results indicate that COX-2-selective inhibitors can be used as an alternative treatment to conventional NSAID, especially in situations in which risk factors for nephrotoxicity are present.

The bradycardic and hypotensive responses to serotonin are reduced by activation of GABA A receptors in the nucleus tractus solitarius of awake rats

We investigated the effects of bilateral injections of the GABA receptor agonists muscimol (GABA A) and baclofen (GABA B) into the nucleus tractus solitarius (NTS) on the bradycardia and hypotension induced by iv serotonin injections (5-HT, 2 µg/rat) in awake male Holtzman rats. 5-HT was injected in rats with stainless steel cannulas implanted bilaterally in the NTS, before and 5, 15, and 60 min after bilateral injections of muscimol or baclofen into the NTS. The responses to 5-HT were tested before and after the injection of atropine methyl bromide. Muscimol (50 pmol/50 nl, N = 8) into the NTS increased basal mean arterial pressure (MAP) from 115 ± 4 to 144 ± 6 mmHg, did not change basal heart rate (HR) and reduced the bradycardia (-40 ± 14 and -73 ± 26 bpm at 5 and 15 min, respectively, vs -180 ± 20 bpm for the control) and hypotension (-11 ± 4 and -14 ± 4 mmHg, vs -40 ± 9 mmHg for the control) elicited by 5-HT. Baclofen (12.5 pmol/50 nl, N = 7) into the NTS also increased basal MAP, but did not change basal HR, bradycardia or hypotension in response to 5-HT injections. Atropine methyl bromide (1 mg/kg body weight) injected iv reduced the bradycardic and hypotensive responses to 5-HT injections. The stimulation of GABA A receptors in the NTS of awake rats elicits a significant increase in basal MAP and decreases the cardiac Bezold-Jarisch reflex responses to iv 5-HT injections.

Perspectives of digestive pest control with proteinase inhibitors that mainly affect the trypsin-like activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)

The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4ºC and the supernatants were used in enzymatic assays at 30ºC, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis) was 1.14 ± 0.15 absorbance variation min-1 mg protein-1, at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis) was 0.217 ± 0.02 mmol p-nitroaniline min-1 mg protein-1. The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50ºC, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.