Expression of stress-related genes in diapause of European corn borer (Ostrinia nubilalis Hbn.).

Diapause is a state of arrested development during which insects cope with many external and internal stressful factors. European corn borer, Ostrinia nubilalis, overwinters as a fifth instar freeze-tolerant diapausing larva. In order to explore diapause-linked stress tolerance processes, the expression of selected genes coding for stress-related proteins-glutathione S-transferase (Gst), thioredoxin (Trx), glutaredoxin (Grx), ferritin (Fer), metallothionein (Mtn), and heat shock proteins Hsp90, Hsc70, Hsp20.4, and Hsp20.1-was assessed in the fat body of diapause-destined, warm (22 °C) and cold (5 °C) acclimated diapausing larvae using the quantitative real-time PCR. Gene expression was normalised to mRNA transcripts for Actin and Rps03, and relative expression was calculated using non-diapausing larvae as a control group. During the initiation phase of diapause, the abundance of mRNA transcripts of Grx, Hsp90, Hsc70, and Hsp20.1 was significantly upregulated, Trx, Fer, Mtn, and Hsp20.1 were unchanged, while only Gst was clearly downregulated in comparison to non-diapause control. Later, in the early phase of diapause, the expression of most genes (except Trx and Hsp20.1) was upregulated in warm-acclimated larvae, while only Trx and Hsp90 were upregulated in cold-acclimated larvae. Furthermore, the relative expression of all genes (except Trx) increased gradually throughout the diapause in cold-acclimated larvae. This result indicates that the half-life of mRNAs is prolonged during diapause at low temperature, which may lead to a gradual accumulation of mRNA transcripts. Our results show that both diapause programming and temperatures affect the expression of stress-related genes in Ostrinia nubilalis.

Reversible oxidation of myometrial voltage-gated potassium channels with hydrogen peroxide.

The uteri, spontaneously active or Ca(2+) (6 mM) induced, were allowed to equilibrate, and to inhibit voltage-gated potassium (K(V)) channels 1 mM 4-amino pyridine (4-AP) was applied for 15 min before adding H(2)O(2). H(2)O(2) was added cumulatively: 2 µM, 20 µM, 200 µM, 400 µM, and 3 mM. Average time for H(2)O(2) concentrations (2, 20, 200, and 400) µM to reach its full effect was 15 min. H(2)O(2) 3 mM had a prolonged effect and therefore was left to act for 30 min. Two-way ANOVA showed significant differences in time dependency between spontaneous and Ca(2+)-induced rat uteri after applying 3 mM H(2)O(2) (type of contraction, P = 0.0280), but not 400 µM H(2)O(2) (P = 0.9271). Our results indicate that H(2)O(2) oxidises channel intracellular thiol groups and activates the channel, inducing relaxation. Cell antioxidative defence system quickly activates glutathione peroxidase (GSHPx) defence mechanism but not catalase (CAT) defence mechanism. Intracellular redox mechanisms repair the oxidised sites and again establish deactivation of K(V) channels, recuperating contractility. In conclusion, our results demonstrate that K(V) channels can be altered in a time-dependent manner by reversible redox-dependent intracellular alterations.

Effect of indometacin pretreatment on protamine sulfate-mediated relaxation of the isolated rat uterus: the role of the antioxidative defense system.

Previous results in this laboratory indicate that protamine sulfate (PS) evokes dose-dependent relaxation of both spontaneous and calcium ion-induced uterus activity mediated predominantly by potassium channels and, to a small extent, via ß-adrenergic receptors or nitric oxide (NO)-dependent pathways. Indometacin is a nonselective inhibitor of cyclooxygenase (COX 1 and COX 2) that has the ability to delay premature labor by reducing uterine contractions through the inhibition of prostanglandin synthesis in the uterus. This study investigates the effects of indometacin (0.1 and 1 µg/ml) pretreatment on the PS-induced relaxation of isolated uterine smooth muscle. Indometacin pretreatment per se did not change the activity of the uteri. However, indometacin significantly increased PS-induced relaxation of spontaneous uterine contractions. Indometacin pretreatment significantly decreased the magnitude and slope of PS-induced relaxation of calcium ion-induced uterine contractions. Indometacin pretreatment increased CuZnSOD activity and slightly increased GR activity during spontaneous uterine contractions when compared to PS alone. In calcium ion-induced contractions, indometacin pretreatment increased CuZnSOD, GSH-Px and GR activities. These results suggest that, in addition to its COX inhibitory effects, indometacin influences the effects of PS. Therefore, it is possible that indometacin regulates diverse cell functions via its association with lipid membranes by altering micro-environments within the membranes. The above-mentioned processes appear to be partly mediated by redox processes involving ROS, lipid peroxides and antioxidant enzymes. The extent of the PS-mediated effect as different in spontaneous versus calcium ion-induced active uteri.

Effects of cadmium on protocerebral neurosecretory neurons and fitness components in Lymantria dispar L.

Changes in fitness components including larval stage duration, relative growth rate (RGR), and mass of the gypsy moth, Lymantria dispar L. (Lepidoptera: Lymantriidae), were investigated in caterpillars fed a synthetic diet with or without a cadmium supplement (10, 30, 100, 250 microg Cd/g dry food weight). Morphometric changes of large protocerebral dorsomedial A2 neurosecretor neurons, their nuclei and the electrophoresis profiles of brain proteins were analyzed in the 4 instar gypsy moths fed the examined diets. The duration of the fourth larval instars were prolonged and RGR and body mass reduced if the caterpillars were fed diets containing high concentrations of cadmium (100 and 250 microg). The size of large A2 dorsomedial neurosecretory neurons and their nuclei were significantly higher in larvae fed the diets supplemented with 10, 100 and 250 microg Cd. A large amount of neurosecretory material appeared in dorsomedial neurosecretory neurons in larvae fed diets with 100 and 250 microg Cd. Differences in larval brain protein profiles in the region of molecular mass ranges (Mr) of 98 kDa, 46 kDa and 3.4-6.1 kDa were identified in the experimental groups.

Effects of protamine sulphate on spontaneous and calcium-induced contractile activity in the rat uterus are potassium channels-mediated.

Protamine sulphate (PS) effect on spontaneous and calcium-induced rhythmic contractions of isolated virgin rat uteri was studied. PS caused dose-dependent relaxation of both types of contractions (two-way ANOVA, significant dose effects). Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME; 10(-5) mol/l), methylene blue (MB; 0.9 x 10(-6) mol/l) or propranolol (1.7 x 10(-5) mol/l) enhanced PS-mediated uterine muscle relaxation of spontaneous contractions. Dosedependent relaxation of spontaneous active isolated rat uterus with PS was lower in uteri pretreated with single dose of tetraethylammonium (TEA; 6 x 10(-3) mol/l), glibenclamide (2 x 10(-6) mol/l) and 4-aminopyridine (4-AP; 10(-3) mol/l). Calcium-induced activity of the isolated rat uterus pretreated with the same concentration of L-NAME, MB, or propranolol modified the kinetic of PS-induced relaxation without changes in EC(50) values. Pre-treatment with glibenclamide, TEA and 4-AP significantly reduce PS relaxing effect of calcium-induced activity and according to EC(50) values the order of magnitude was glibenclamide > TEA > 4-AP. PS is mixture of polyamines and may activate different signal-transduction pathways. Our results cleary demonstrate that in uterine smooth muscle PS act dominantly through potassium chanels and marginaly through beta-adrenergic receptos or nitric oxide-dependent pathways.

Diethyldithiocarbamate potentiates the effects of protamine sulphate in the isolated rat uterus.

Protamine sulphate causes potassium ion channel-mediated relaxation of spontaneous and calcium ion-induced contractions of the isolated rat uterus. Diethyldithiocarbamate (DDC) potentiated the effect of protamine sulphate. A mechanism for DDC's action was postulated on the basis of its interactions with divalent iron ions and Cu,Zn-SOD. DDC chelates divalent iron ions creating DDC-iron (Fe-DDC) complexes. Fe-DDC forms stable NO-Fe-DDC(2) complexes by NO scavenging and de-nitrosylation processes, which in combination with DDC (5 mM) provoke inhibition of Cu,Zn-SOD resulting in specific oxidative conditions culminating in potassium ion channel opening, membrane hyperpolarisation, inhibition of calcium ion influx and subsequent muscle relaxation. As Fe-DDC and NO-Fe-DDC(2) complexes exclude divalent iron ions from participating in the hydroxyl radical generating Fenton reaction, DDC can also prevent iron-related pathophysiological manifestations. Such permissive roles of DDC open the possibility for application of its pharmacological form (disulfiram) to a wider spectrum of pathophysiological conditions related to smooth muscles.

Superoxide dismutase and lipid hydroperoxides in blood and endometrial tissue of patients with benign, hyperplastic and malignant endometrium.

Epidemiological and experimental data point to involvement of oxygen derived radicals in the pathogenesis of gynecological disorders, as well as in cancer development. The objective of the present study was to examine changes in activities and levels of copper/zinc superoxide dismutase (CuZnSOD) and lipid hydroperoxides (LOOH) in blood and endometrial tissue of patients diagnosed with uterine myoma, endometrial polypus, hyperplasia simplex, hyperplasia complex and adenocarcinoma endometrii. The results of our study have shown decreased SOD activities and unchanged SOD protein level in blood of all examined patients in comparison to healthy subjects. Decrease of both SOD activity and level was found in endometrium of patients with hyperplasia simplex, hyperplasia complex and adenocarcinoma in comparison to women with polypus or myoma. LOOH level was elevated in both tissues of patients with hyperplasia or adenocarcinoma in comparison to healthy subjects or patients with benign diagnosis. Our findings suggest that the decrease in SOD activity and level, as well as the increase in LOOH level, in patients with gynecological disorders, render these patients more susceptible to oxidative damage caused by reactive oxygen species (ROS). An imbalance in ROS formation and SOD level may be important in the pathogenesis and/or perpetuation of tissue damage in gynecological patients. Since evidence suggests that SOD may be a therapy target for cancer treatment, our findings provide a basis for further research and options for clinical applications.

Superoxide dismutase and lipid hydroperoxides in blood and endometrial tissue of patients with benign, hyperplastic and malignant endometrium

Epidemiological and experimental data point to involvement of oxygen derived radicals in the pathogenesis of gynecological disorders, as well as in cancer development. The objective of the present study was to examine changes in activities and levels of copper/zinc superoxide dismutase (CuZnSOD) and lipid hydroperoxides (LOOH) in blood and endometrial tissue of patients diagnosed with uterine myoma, endometrial polypus, hyperplasia simplex, hyperplasia complex and adenocarcinoma endometrii. The results of our study have shown decreased SOD activities and unchanged SOD protein level in blood of all examined patients in comparison to healthy subjects. Decrease of both SOD activity and level was found in endometrium of patients with hyperplasia simplex, hyperplasia complex and adenocarcinoma in comparison to women with polypus or myoma. LOOH level was elevated in both tissues of patients with hyperplasiaor adenocarcinoma in comparison to healthy subjects or patients with benign diagnosis. Our findings suggest that the decrease in SOD activity and level, as well as the increase in LOOH level, in patients with gynecological disorders, render these patients more susceptible to oxidative damage caused by reactive oxygen species (ROS). An imbalance in ROS formation and SOD level may be important in the pathogenesis and/or perpetuation of tissue damage in gynecological patients. Since evidence suggests that SOD may be a therapy target for cancer treatment, our findings provide a basis for further research and options for clinical applications.

Resultados epidemiológicos e experimentais apontam para o envolvimento dos radicais derivados do oxigênio na patogênese das moléstias ginecológicas, assim como no desenvolvimento do câncer. O objetivo do presente estudo foi o de examinar as alterações nas atividades e níveis de Cu/Zn superóxido dismutase (CuZnSOD) e hidroperóxidos lipídicos (LOOH)no sangue e tecido endometrial de pacientes diagnosticados com mioma uterino, pólipo endometrial, hiperplasia simplex, hiperplasia complex e adenocarcinoma do endométrio. Os resultados de nosso estudo mostraram atividades de SOD diminuídas e nível de SOD proteína inalterado no sangue de todos os pacientes examinados em comparação a indivíduos saudáveis. Diminuição de ambos, atividade de SOD e nível protéico, foram encontrados no endométrio de pacientes com hiperplasia simplex, hiperplasia complex e adenocarcinoma em comparação às mulheres com pólipos e/ou mioma. O nível de LOOH estava elevado em ambos os tecidos de pacientes com hyperplasia e adenocarcinoma em comparação a indivíduos saudáveis ou pacientes com diagnóstico benigno. Nossos resultados sugerem que um decréscimo na atividade e nível protéico de SOD, assim como um incremento no nível de LOOH, em pacientes com desordens ginecológicas, tornam esses pacientes mais susceptíveis ao dano oxidativo causado pelas espécies reativas de oxigênio (ROS). Um desequilíbrio na formação de ROS e no nível de SOD pode ser importante na patogênese e/ou perpetuação do dano tecidual em pacientes ginecológicos. Desde que existe evidência de que SOD pode ser um alvo para terapia de câncer, nossos resultados fornecem uma base para futura pesquisa e opções para aplicações clínicas.

A comparative study of the effects of molsidomine and 3-morpholinosydnonimine on the redox status of rat erythrocytes and reticulocytes.

After enzymic biotransformation, molsidomine (MO) acts via the metabolite 3-morpholinosydnonimine (SIN-1) through spontaneous liberation of nitric oxide (NO) and superoxide (O(2)(.-)). The aim of this study was to compare the effects of MO and its active metabolite SIN-1 on the redox status of rat erythrocytes and reticulocytes. Rat erythrocyte as well as reticulocyte-rich red blood cell (RBC) suspensions were aerobically incubated (2 h, 37 degrees C) without (control) or in the presence of different concentrations of MO or SIN-1. In rat erythrocytes, biotransformation of MO resulted in the production of NO and nitroxyl (NO(-)). Endogenous superoxide anion (O(2)(.-)) participated in peroxynitrite generation. SIN-1 simultaneously liberated NO and O(2)(.-), which formed peroxynitrite (at least in part), but the liberated NO predominantly reacted with haemoglobin, forming methaemoglobin in erythrocytes. In reticulocytes, MO and SIN-1 caused an increase in the levels of both nitrite and 3-nitrotyrosine (an indicator of peroxynitrite), whereas they decreased the level of O(2)(.-). In reticulocytes, MO was metabolized into SIN-1 which led to the generation of NO, which reacted with O(2)(.-) (endogenous or exogenous) forming reactive nitrogen species. In conclusion, there are two metabolic pathways for MO biotransformation: one causing NO and NO(-) generation predominantly in erythrocytes and the other, via SIN-1 metabolism, in reticulocytes. The main difference between the action of MO and SIN-1 was that the latter caused oxidative damage in RBCs.

The activity of antioxidant defence enzymes in the mussel Mytilus galloprovincialis from the Adriatic Sea.

The activity of the antioxidant defence enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), glutathione peroxidase (GSH-Px, EC 1.11.1.9), glutathione reductase (GR, EC 1.6.4.2) and the phase II biotransformation enzyme glutathione-S-transferase (GST, EC 2.5.1.18) in whole mussels (Mytilus galloprovincialis) were studied. The mussels were collected in winter and in spring at two localities in the Adriatic Sea: Bar Port and Tivat Bay. Our results show that the activities of SOD, GSH-Px and GST were seasonally dependent with higher activities in winter. GR activity was also higher in winter, but only in mussels from Bar Port. In mussels from Tivat Bay, GR activity was lower in winter compared to spring. In addition, a decrease in CAT activity in mussels from Bar Port compared to those from Tivat Bay was found. It can be concluded that seasonal variations should be incorporated into interpretation of biomonitoring studies in mussels.

Seasonal variations of cytosolic antioxidant enzyme activities in the liver and white muscle of thinlip gray mullet (Liza ramada Risso) from the Adriatic Sea.

The activity of cytosolic antioxidative defence enzymes in the liver and white muscle of thinlip gray mullet (Liza ramada Risso) were compared in winter and spring in the Adriatic Sea. Activity of antioxidative enzymes is functionally organized due to metabolic demands: analyses of variance and correlation analysis revealed tissue- and seasonal- specific organization of antioxidative enzymes. In winter GST activity increased in both tissues compared with spring. At the same time decreased GSH-Px and GR activities were observed and this effect was more pronounced in liver then in white muscle. From correlation analyses it is concluded that the antioxidative components correlate, but the composition of the antioxidative defence system is different in respect to season and tissue. This means that the antioxidative defence system reorganizes its structure due to oxidative demands and to protect the tissues against reactive oxygen species and to establish homeostasis. Discriminant analyses separated groups according to the complete organization of individual components of the system very well and identified individual components (CAT, GST and GR) which contribute most to the differences. Statistical differences were observed between enzyme activities in tissues (liver and muscle) in both winter and spring, and between seasons (winter and spring) for liver tissue only. Since environmental parameters, such as temperature and oxygen concentration in the sea differ with season, we conclude that in this species the tissues examined expressed their antioxidative defence systems in different ways in respect of external/environmental conditions. We propose that tissue- and seasonal- specific levels of antioxidant enzyme activities should be considered in the interpretation of data from future biomonitoring field studies, especially in relation to low temperature.