Low pH and calcium effects on net Na+ and K+ fluxes in two catfish species from the Amazon River (Corydoras: Callichthyidae)

The present study analyzes Na+ and K+ disturbances caused by low pH in two catfish species from the Amazon River. Corydoras adolfoi inhabits ion-poor, black-stained, low pH (3.5-4.0) waters, while C. schwartzi is native to ion-rich waters at circumneutral pH. Fish were exposed to pH 3.5 Ca2+-free, and Ca2+-enriched (~500 µmol/l) water to determine the protective effects of calcium. Net Na+ and K+ fluxes were measured in the water collected from the fish experimental chambers. C. adolfoi was unable to control the Na+ efflux at low pH, exhibiting Na+ loss up to -594 ± 84 nmol g-1 h-1 during the first hour. After 3 and 6 h, net Na+ flux increased by 7- and 23-fold, respectively. In C. schwartzi, at pH 3.5, the initial high Na+ loss (-1,063 ± 73 nmol g-1 h-1) was gradually attenuated. A K+ loss occurred in both species, but remained relatively constant throughout exposure. High [Ca2+] affected ion losses in both species. C. adolfoi had 70% loss attenuation, indicating incapacity to control Na+ efflux. In C. schwartzi, elevated [Ca2+] completely prevented the Na+ losses caused by exposure to low pH. Rather different patterns were seen for K+ fluxes, with C. adolfoi showing no K+ disruption when exposed to low pH/high [Ca2+]. Thus, C. adolfoi loses Na+ during acid exposure, but has the ability to control K+ loss, while C. schwartzi controls diffusive Na+ loss but exhibits a slightly higher K+ loss. Ion balance was influenced by [Ca2+] at low pH in C. schwartzi but not in C. adolfoi.

Effect of hydroxyurea on the intracellular multiplication of Toxoplasma gondii, Leishmania amazonensis and Trypanosoma cruzi

Toxoplasma gondii, Leishmania amazonensis and Trypanosoma cruzi are obligate intracellular parasites that multiply until lysis of host cells. The present study was undertaken to evaluate the effect of hydroxyurea (an inhibitor of cell division at the G1/S phase) on the multiplication of L. amazonensis, T. gondii, and T. cruzi in infected host cells. Infected cells were treated with hydroxyurea (4 mM) for 48 h. Hydroxyurea arrested intracellular multiplication of all infective forms of the parasites tested. In treated cultures, the percent of infected host cells decreased (50-97%) and most intracellular parasites were eliminated. Ultrastructural observations showed no morphologic change in host cells while intracellular parasites presented drastic morphologic alterations or disruption. The results strongly suggest that hydroxyurea was able to interfere with the multiplication of intracellular parasites, leading to an irreversible morphological effect on L. amazonensis, T. gondii, and T. cruzi without affecting the host cells.

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.

Routine post-weaning handling of rats prevents isolation rearing-induced deficit in prepulse inhibition

Rats reared under isolation conditions from weaning present a number of behavioral changes compared to animals reared under social conditions (group housing). These changes include deficits in prepulse inhibition (PPI) of the startle reflex to a loud sound. PPI refers to the reduction of the magnitude of the startle reflex when a relatively weak stimulus (the prepulse) precedes by an appropriate time interval the intense startle-elicing stimulus (the pulse). PPI is useful for studying sensorimotor integration. The present study evaluated the effect of handling on the impairment of PPI induced by isolation-rearing. Male Wistar rats (N = 11-15/group) were housed in groups (5 per cage and handled three times a week) or isolated (housed individually) since weaning (21 days) for 10 weeks when they reach approximately 150 g. The isolated rats were divided into "minimally handled" animals (handled once a week for cleaning purposes only) or "handled" animals (handled three times a week). This handling consisted of grasping the rat by the tail and moving it to a clean cage (approximately 5 s). A statistically significant reduction (52%) in the PPI test was found only in the isolated group with minimal handling while no difference was seen between grouped animals and isolated handled animals. These results indicate that isolation rearing causes disruption in the PPI at adult age, which serves as an index of attention deficit. This change in the sensory processing of information induced by post-weaning isolation can be prevented by handling during the development of the animal.

Liver mitochondrial dysfunction and oxidative stress in the pathogenesis of experimental nonalcoholic fatty liver disease

Oxidative stress and hepatic mitochondria play a role in the pathogenesis of nonalcoholic fatty liver disease. The aim of the present study was to evaluate the role of hepatic mitochondrial dysfunction and oxidative stress in the pathogenesis of the disease. Fatty liver was induced in Wistar rats with a choline-deficient diet (CD; N = 7) or a high-fat diet enriched with PUFAs-omega-3 (H; N = 7) for 4 weeks. The control group (N = 7) was fed a standard diet. Liver mitochondrial oxidation and phosphorylation were measured polarographically and oxidative stress was estimated on the basis of malondialdehyde and glutathione concentrations. Moderate macrovacuolar liver steatosis was observed in the CD group and mild liver steatosis was observed in the periportal area in the H group. There was an increase in the oxygen consumption rate by liver mitochondria in respiratory state 4 (S4) and a decrease in respiratory control rate (RCR) in the CD group (S4: 32.70 ± 3.35; RCR: 2.55 ± 0.15 ng atoms of O2 min-1 mg protein-1) when compared to the H and control groups (S4: 23.09 ± 1.53, 17.04 ± 2.03, RCR: 3.15 ± 0.15, 3.68 ± 0.15 ng atoms of O2 min-1 mg protein-1, respectively), P < 0.05. Hepatic lipoperoxide concentrations were significantly increased and the concentration of reduced glutathione was significantly reduced in the CD group. A choline-deficient diet causes moderate steatosis with disruption of liver mitochondrial function and increased oxidative stress. These data suggest that lipid peroxidation products can impair the flow of electrons along the respiratory chain, causing overreduction of respiratory chain components and enhanced mitochondrial reactive oxygen species. These findings are important in the pathogenesis of nonalcoholic fatty liver disease.

Contested dominance modifies the anovulatory consequences of social subordination in female marmosets

Dominance status among female marmosets is reflected in agonistic behavior and ovarian function. Socially dominant females receive submissive behavior from subordinates, while exhibiting normal ovulatory function. Subordinate females, however, receive agonistic behavior from dominants, while exhibiting reduced or absent ovulatory function. Such disparity in female fertility is not absolute, and groups with two breeding females have been described. The data reported here were obtained from 8 female-female pairs of captive female marmosets, each housed with a single unrelated male. Pairs were classified into two groups: "uncontested" dominance (UD) and "contested" dominance (CD), with 4 pairs each. Dominant females in UD pairs showed significantly higher frequencies (4.1) of agonism (piloerection, attack and chasing) than their subordinates (0.36), and agonistic behaviors were overall more frequently displayed by CD than by UD pairs. Subordinates in CD pairs exhibited more agonistic behavior (2.9) than subordinates in UD pairs (0.36), which displayed significantly more submissive (6.97) behaviors than their dominants (0.35). The data suggest that there is more than one kind of dominance relationship between female common marmosets. Assessment of progesterone levels showed that while subordinates in UD pairs appeared to be anovulatory, the degree of ovulatory disruption in subordinates of CD pairs was more varied and less complete. We suggest that such variation in female-female social dominance relationships and the associated variation in the degree and reliability of fertility suppression may explain variations of the reproductive condition of free-living groups of common marmosets.

Gastroesophageal reflux episodes in asthmatic patients and their temporal relation with sleep architecture

Gastroesophageal reflux (GER) is common in asthma patients and can contribute to sleep disruption. The aim of the present study was to determine the time-related distribution of GER events together with their impact on sleep in asthmatic subjects with GER disease symptoms. The inclusion criteria were: 18-65 years, controlled moderate to severe asthma and GER-compatible clinical evidence. The exclusion criteria were: chronic obstructive lung disease, smoking, infections of the upper airways, use of oral corticosteroids, other co-morbidities, pregnancy, sleep-related disorders, night-time shift work, and the use of substances with impact on sleep. Asthmatic patients with nocturnal symptoms were excluded. All-night polysomnography and esophageal pH monitoring were recorded simultaneously. Of the 147 subjects selected, 31 patients and 31 controls were included. Seventeen patients were classified as DeMeester positive and 14 as DeMeester negative. Both groups displayed similar outcomes when general variables were considered. Sleep stage modification one minute prior to GER was observed in the DeMeester-positive group. Awakening was the most frequent occurrence at GER onset and during the 1-min period preceding 38% of the nocturnal GER. Sleep stage 2 was also prevalent and preceded 36% of GER events. In the DeMeester-negative group, awakening was the most frequent response before and during GER. Modifications in sleep stages, arousals or awakenings were associated with 75% of the total GER events analyzed during the period of one minute before and after the fall of esophageal pH below 4 in the DeMeester-positive group. These data provide evidence that sleep modifications precede the GER events in asthmatic patients.

Solubilization of human erythrocyte membranes by ASB detergents

Understanding the membrane solubilization process and finding effective solubilizing agents are crucial challenges in biochemical research. Here we report results on the interaction of the novel linear alkylamido propyl dimethyl amino propanosulfonate detergents, ASB-14 and ASB-16, with human erythrocyte membranes. An estimation of the critical micelle concentration of these zwitterionic detergents (ASB-14 = 100 µM and ASB-16 = 10 µM) was obtained using electron paramagnetic resonance. The amount of proteins and cholesterol solubilized from erythrocytes by these detergents was then determined. The hemolytic activities of the ASB detergents were assayed and the detergent/lipid molar ratios for the onset of hemolysis (Re sat) and total lysis (Re sol) were calculated, allowing the determination of the membrane binding constants (Kb). ASB-14 presented lower membrane affinity (Kb = 7050 M-1) than ASB-16 (Kb = 15610 M-1). The amount of proteins and cholesterol solubilized by both ASB detergents was higher while Re sat values (0.22 and 0.08 detergent/lipid for ASB-14 and ASB-16, respectively) were smaller than those observed with the classic detergents CHAPS and Triton X-100. These results reveal that, besides their well-known use as membrane protein solubilizers to enhance the resolution of two dimensional electrophoresis/mass spectrometry, ASB-14 and ASB-16 are strong hemolytic agents. We propose that the physicochemical properties of ASB detergents determine their membrane disruption efficiency and can help to explain the improvement in the solubilization of membrane proteins, as reported in the literature.

Delta sleep instability in children with chronic arthritis

The objective of the present study was to evaluate the expression of a cyclic alternating pattern (CAP) in slow wave sleep (SWS) in children with the well-defined chronic syndrome juvenile idiopathic arthritis (JIA). Twelve patients (9-17 years of age), 7 girls, with JIA were compared to matched controls by age, pubertal stage and gender. After one night of habituation in the sleep laboratory, sleep measurements were obtained by standard polysomnography with conventional sleep scoring and additional CAP analyses. The sleep parameters of the JIA and control groups were similar for sleep efficiency (91.1 ± 6.7 vs 95.8 ± 4.0), sleep stage in minutes: stage 1 (16.8 ± 8.5 vs 17.8 ± 4.0), stage 2 (251.9 ± 41 vs 262.8 ± 38.1), stage 3 (17.0 ± 6.0 vs 15.1 ± 5.7), stage 4 (61.0 ± 21.7 vs 77.1 ± 20.4), and rapid eye movement sleep (82.0 ± 27.6 vs 99.0 ± 23.9), respectively. JIA patients presented nocturnal disrupted sleep, with an increase in short awakenings, but CAP analyses showed that sleep disruption was present even during SWS, showing an increase in the overall CAP rate (P < 0.01). Overall CAP rate during non-rapid eye movement sleep was significantly higher in pediatric patients who were in chronic pain. This is the first study of CAP in pediatric patients with chronic arthritis showing that CAP analyses can be a powerful tool for the investigation of disturbance of SWS in children, based on sleep EEG visual analysis.

Potential pathogenic role of aggregative- adhering Corynebacterium diphtheriae of different clonal groups in endocarditis

Invasive diseases caused by Corynebacterium diphtheriae have been described increasingly. Several reports indicate the destructive feature of endocarditis attributable to nontoxigenic strains. However, few reports have dealt with the pathogenicity of invasive strains. The present investigation demonstrates a phenotypic trait that may be used to identify potentially invasive strains. The study also draws attention to clinical and microbiological aspects observed in 5 cases of endocarditis due to C. diphtheriae that occurred outside Europe. Four cases occurred in female school-age children (7-14 years) treated at different hospitals in Rio de Janeiro, Brazil. All patients developed other complications including septicemia, renal failure and/or arthritis. Surgical treatment was performed on 2 patients for valve replacement. Lethality was observed in 40% of the cases. Microorganisms isolated from 5 blood samples and identified as C. diphtheriae subsp mitis (N = 4) and C. diphtheriae subsp gravis (N = 1) displayed an aggregative adherence pattern to HEp-2 cells and identical one-dimensional SDS-PAGE protein profiles. Aggregative-adhering invasive strains of C. diphtheriae showed 5 distinct RAPD profiles. Despite the clonal diversity, all 5 C. diphtheriae invasive isolates seemed to display special bacterial adhesive properties that may favor blood-barrier disruption and systemic dissemination of bacteria. In conclusion, blood isolates from patients with endocarditis exhibited a unique adhering pattern, suggesting a pathogenic role of aggregative-adhering C. diphtheriae of different clones in endocarditis. Accordingly, the aggregative-adherence pattern may be used as an indication of some invasive potential of C. diphtheriae strains.

Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of Streptococcus mutans

Streptococcus mutans membrane-bound P- and F-type ATPases are responsible for H+ extrusion from the cytoplasm thus keeping intracellular pH appropriate for cell metabolism. Toluene-permeabilized bacterial cells have long been used to study total membrane-bound ATPase activity, and to compare the properties of ATPase in situ with those in membrane-rich fractions. The aim of the present research was to determine if toluene permeabilization can significantly modify the activity of membrane-bound ATPase of both F-type and P-type. ATPase activity was assayed discontinuously by measuring phosphate release from ATP as substrate. Treatment of S. mutans membrane fractions with toluene reduced total ATPase activity by approximately 80% and did not allow differentiation between F- and P-type ATPase activities by use of the standard inhibitors vanadate (3 µM) and oligomycin (4 µg/mL). Transmission electron microscopy shows that, after S. mutans cells permeabilization with toluene, bacterial cell wall and plasma membrane are severely injured, causing cytoplasmic leakage. As a consequence, loss of cell viability and disruption of H+ extrusion were observed. These data suggest that treatment of S. mutans with toluene is an efficient method for cell disruption, but care should be taken in the interpretation of ATPase activity when toluene-permeabilized cells are used, because results may not reflect the real P- and F-type ATPase activities present in intact cell membranes. The mild conditions used for the preparation of membrane fractions may be more suitable to study specific ATPase activity in the presence of biological agents, since this method preserves ATPase selectivity for standard inhibitors.

Anti-parasitic action and elimination of intracellular Toxoplasma gondii in the presence of novel thiosemicarbazone and its 4-thiazolidinone derivatives

Toxoplasma, which infects all eukaryotic cells, is considered to be a good system for the study of drug action and of the behavior of infected host cells. In the present study, we asked if thiosemicarbazone derivatives can be effective against tachyzoites and which morphological and ultrastructural features of host cells and parasites are associated with the destruction of Toxoplasma. The compounds were tested in infected Vero cell culture using concentration screens (0.1 to 20 mM). The final concentration of 1 mM was chosen for biological assay. The following results were obtained: 1) These new derivatives decreased T. gondii infection with an in vitro parasite IC50% of 0.2-0.7 mM, without a significant effect on host cells and the more efficient compounds were 2, 3 (thiosemicarbazone derivatives) and 4 (thiazolidinone derivative); 2) The main feature observed during parasite elimination was continuous morphological disorganization of the tachyzoite secretory system, progressive organelle vesiculation, and then complete disruption; 3) Ultrastructural assays also revealed that progressive vesiculation in the cytoplasm of treated parasites did not occur in the host cell; 4) Vesiculation inside the parasite resulted in death, but this feature occurred asynchronously in different intracellular tachyzoites; 5) The death and elimination of T. gondii was associated with features such as apoptosis-like stage, acidification and digestion of parasites into parasitophorous vacuoles. Our results suggest that these new chemical compounds are promising for the elimination of intracellular parasites by mainly affecting tachyzoite development at 1 mM concentration for 24 h of treatment.

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

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

Protective effects of organoselenium compounds against methylmercury-induced oxidative stress in mouse brain mitochondrial-enriched fractions

We evaluated the potential neuroprotective effect of 1-100 µM of four organoselenium compounds: diphenyl diselenide, 3’3-ditri-fluoromethyldiphenyl diselenide, p-methoxy-diphenyl diselenide, and p-chloro-diphenyl diselenide, against methylmercury-induced mitochondrial dysfunction and oxidative stress in mitochondrial-enriched fractions from adult Swiss mouse brain. Methylmercury (10-100 µM) significantly decreased mitochondrial activity, assessed by MTT reduction assay, in a dose-dependent manner, which occurred in parallel with increased glutathione oxidation, hydroperoxide formation (xylenol orange assay) and lipid peroxidation end-products (thiobarbituric acid reactive substances, TBARS). The co-incubation with diphenyl diselenide (100 µM) completely prevented the disruption of mitochondrial activity as well as the increase in TBARS levels caused by methylmercury. The compound 3’3-ditrifluoromethyldiphenyl diselenide provided a partial but significant protection against methylmercury-induced mitochondrial dysfunction (45.4 ± 5.8% inhibition of the methylmercury effect). Diphenyl diselenide showed a higher thiol peroxidase activity compared to the other three compounds. Catalase blocked methylmercury-induced TBARS, pointing to hydrogen peroxide as a vector during methylmercury toxicity in this model. This result also suggests that thiol peroxidase activity of organoselenium compounds accounts for their protective actions against methylmercury-induced oxidative stress. Our results show that diphenyl diselenide and potentially other organoselenium compounds may represent important molecules in the search for an improved therapy against the deleterious effects of methylmercury as well as other mercury compounds.

The influence of stress on fear memory processes

It is well recognized that stressful experiences promote robust emotional memories, which are well remembered. The amygdaloid complex, principally the basolateral complex (BLA), plays a pivotal role in fear memory and in the modulation of stress-induced emotional responses. A large number of reports have revealed that GABAergic interneurons provide a powerful inhibitory control of the activity of projecting glutamatergic neurons in the BLA. Indeed, a reduced GABAergic control in the BLA is essential for the stress-induced influence on the emergence of associative fear memory and on the generation of long-term potentiation (LTP) in BLA neurons. The extracellular signal-regulated kinase (ERK) subfamily of the mitogen-activated protein kinase (MAPK) signaling pathway in the BLA plays a central role in the consolidation process and synaptic plasticity. In support of the view that stress facilitates long-term fear memory, stressed animals exhibited a phospho-ERK2 (pERK2) increase in the BLA, suggesting the involvement of this mechanism in the promoting influence of threatening stimuli on the consolidation fear memory. Moreover, the occurrence of reactivation-induced lability is prevented when fear memory is encoded under intense stressful conditions since the memory trace remains immune to disruption after recall in previously stressed animals. Thus, the underlying mechanism in retrieval-induced instability seems not to be functional in memories formed under stress. All these findings are indicative that stress influences both the consolidation and reconsolidation fear memory processes. Thus, it seems reasonable to propose that the emotional state generated by an environmental challenge critically modulates the formation and maintenance of long-term fear memory.