In Vitro Antifungal Activity and Toxicity of Itraconazole in DMSA-PLGA Nanoparticles

Itraconazole (ITZ) is a drug used to treat various fungal infections and may cause side effects. The aim of this study was to develop and evaluate the in vitro activity of DMSA-PLGA nanoparticles loaded with ITZ against Paracoccidioides brasiliensis, as well as their cytotoxicity. Nanoparticles were prepared using the emulsification-evaporation technique and characterized by their encapsulation efficiency, morphology (TEM), size (Nanosight) and charge (zeta potential). Antifungal efficacy in P brasiliensis was determined by minimal inhibition concentration (MIC), and cytotoxicity using MU assay. ITZ was effectively incorporated in the PLGA-DMSA nanoparticles with a loading efficiency of 72.8 +/- 3.50%. The shape was round with a solid polymeric structure, and a size distribution of 174 +/- 86 nm (Average +/- SD). The particles were negatively charged. ITZ-NANO presented antifungal inhibition (MIC = 6.25 ug/mL) against P brasiliensis and showed lower in vitro cytotoxicity than free drug (ITZ).

Shared structural determinants for the calcium-independent liposome membrane permeabilization and sarcolemma depolarization in Bothropstoxin-I, a LYS49-PLA(2) from the venom of Bothrops jararacussu

The structural determinants of myotoxicity of bothropstoxin-I (BthTX-I), a Lys49 phospholipase A(2) from Bothrops jararacussu venom, were studied by measuring the resting membrane potential in the mouse phrenic nerve-diaphragm preparation. This method proved to be around 100-fold more sensitive than the creatine kinase release assay, and was used to evaluate a total of 31 site-directed BthTX-I alanine scanning mutants. Mutants that reduced the resting membrane potential were located in a surface patch defined by residues in the C-terminal loop (residues 115-129), positions 37-39 in the membrane interfacial recognition surface (Y46 and K54), and residue K93. These results expand the known structural determinants of the biological activity as evaluated by previous creatine kinase release experiments. Furthermore, a strong correlation is observed between the structural determinants of sarcolemma depolarization and calcium-independent disruption of liposome membranes, suggesting that a common mechanism of action underlies the permeabilization of the biological and model membranes. (C) 2009 Elsevier Ltd. All rights reserved.

Hemolymph ionic regulation and adjustments in gill (Na(+), K(+))-ATPase activity during salinity acclimation in the swimming crab Callinectes ornatus (Decapoda, Brachyura)

We evaluate hemolymph osmotic and ionic regulatory abilities and characterize a posterior gill microsomal (Na(+), K(+))-ATPase from the marine swimming crab, Callinectes ornatus, acclimated to 21 parts per thousand or 33 parts per thousand salinity. C ornatus is isosmotic after acclimation to 21 parts per thousand but is hyposmotic at 33 parts per thousand salinity; hemolymph ions do not recover initial levels on acclimation to 21 parts per thousand salinity but are anisoionic compared to ambient concentrations, revealing modest regulatory ability. NH(4)(+) modulates enzyme affinity for K(+), which increases 187-fold in crabs acclimated to 33%. salinity. The (Na(+), K(+))-ATPase redistributes into membrane fractions of different densities, suggesting that altered membrane composition results from salinity acclimation. ATP was hydrolyzed at maximum rates of 182.6 +/- 7.1 nmol Pi min(-1) mg(-1) (21 parts per thousand) and 76.2 +/- 3.5 nmol Pi min(-1) mg(-1) (33 parts per thousand), with little change in K(M) values (approximate to 50 mu mol L(-1)). K(+) together with NH(4)(+) synergistically stimulated activity to maximum rates of approximate to 240 nmol Pi min(-1) mg(-1). K, values for ouabain inhibition (approximate to 110 mu mol L(-1)) decreased to 44.9 +/- 1.0 mu mol L(-1) (21 parts per thousand) and 28.8 +/- 1.3 mu mol L(-1) (33 parts per thousand) in the presence of both K(+) and NH(4)(+). Assays employing various inhibitors suggest the presence of mitochondrial F(0)F(1)- and K(+)- and V-ATPase activities in the gill microsomes. (C) 2009 Elsevier Inc. All rights reserved.

Na(+), K(+)-ATPase activity in gill microsomes from the blue crab, Callinectes danae, acclimated to low salinity: Novel perspectives on ammonia excretion

This investigation provides an extensive characterization of the modulation by ATP, Mg(2+), Na(+), K(+) and NH(4)(+) of a gill microsomal (Na(+),K(+))-ATPase from Callinectes danae acclimated to 15 parts per thousand salinity. Novel findings are the lack of high-affinity ATP-binding sites and a 10-fold increase in enzyme affinity for K(+) modulated by NH4+, discussed regarding NH4+ excretion in benthic marine crabs. The (Na(+),K(+))-ATPase hydrolyzed ATP at a maximum rate of 298.7 +/- 16.7 nmol Pi min(-1) mg(-1) and K(0.5) = 174.2 +/- 9.8 mmol L(-1) obeying cooperative kinetics (n(H) = 1.2). Stimulation by sodium (V = 308.9 +/- 15.7 nmol Pi min(-1) mg(-1), K(0.5) = 7.8 +/- 0.4 mmol L(-1)), magnesium (299.2 +/- 14.1 nmol Pi min(-1) mg(-1), K(0.5) = 767.3 +/- 36.1 mmol L(-1)), potassium (300.6 +/- 153 nmol Pi min(-1) mg(-1), K(0.5) = 1.6 +/- 0.08 mmol L(-1)) and ammonium (V = 345.1 +/- 19.0 nmol Pi min(-1) mg(-1), K(0.5) = 6.0 +/- 0.3 mmol L(-1)) ions showed site-site interactions. Ouabain inhibited (Na(+),K(+))-ATPase activity with K(1) = 45.1 +/- 2.5 mu mol L(-1), although affinity for the inhibitor increased (K(1) = 22.7 +/- 1.1 mu mol L(-1)) in 50 mmol L(-1) NH(4)(+). Inhibition assays using ouabain plus oligomycin or ethacrynic acid suggest mitochondrial F(0)F(1)- and K(+)-ATPase activities, respectively. Ammonium and potassium ions synergistically stimulated specific activity up to 72%, inferring that these ions bind to different sites on the enzyme molecule, each modulating stimulation by the other. (C) 2009 Elsevier Inc. All rights reserved.

Activity of the medial prefrontal cortex and amygdala underlies one-trial tolerance of rats in the elevated plus-maze

The anxiolytic effects of benzodiazepines are reduced after a single exposure of rats to elevated plus-maze test (EPM). Midazolam showed an anxioselective profile in animals submitted to one session (T1) but did not change the usual exploratory behavior of rats exposed twice (T2) to the EPM. In this study we examined further the one-trial tolerance by performing a factor analysis of the exploratory behavior of rats injected with saline before both trials as well as an immunohistochemistry study for quantification of Fos expression in encephalic structures after these sessions. Factor analysis of all behavioral categories revealed that factor I consisted of anxiety-related categories in T1 whereas these same behavioral categories loaded on factor 2 in T2. Risk assessment was also dissociated as it loaded stronger on T2 (factor 3) than on T1 (factor 4). Locomotor activity in T1 loaded on factor 5. Immunohistochemistry analyses showed that Fos expression predominated in limbic structures in T1 group. The medial prefrontal cortex and amygdala were the main areas activated in T2 group. These data suggest that anxiety and risk assessment behaviors change their valence across the EPM sessions. T2 is characterized by the emergence of a fear factor, more powerful risk assessment and medial prefrontal cortex activation. The amygdala functions as a switch between the anxiety-like patterns of T1 to the cognitive control of fear prevalent in T2. The EPM retest session is proposed as a tool for assessing the cognitive activity of rodents in the control of fear. (c) 2007 Elsevier B.V. All rights reserved.

Neonatal exposure to LPS leads to heightened exploratory activity in adolescent rats

Although several reports have demonstrated physiological and behavioral changes in adult rats due to neonatal immune challenges, little is known about their effects in adolescence. Since neonatal exposure to lipopolysaccharide (LPS) alters the neural substrates involved in cognitive disorders, we tested the hypothesis that it may also alter the response to novel environments in adolescent rats. At 3 and 5 days of age, male Wistar rats received intraperitoneal injections of either vehicle solution or E. coli LPS (0.05 mg/kg) or were left undisturbed. In the mid-adolescent period, between 40 and 46 days of age, the rats were exposed to the following behavioral tests: elevated plus-maze, open-field, novel-object exploration task, hole-board and the modified Porsolt forced swim test. The results showed that, in comparison with control animals, LPS-treated rats exhibited (1) less anxiety-related behaviors and enhanced patterns of locomotion and rearing in the plus-maze and the open-field tests, (2) high levels of exploration of both objects in the novel-object task and of corner and central holes in hole-board test, and (3) more time spent diving, an active behavior in the forced swim test. The present findings suggest that neonatal LPS exposure has long-lasting effects on the behavior profile adolescent rats exhibit in response to novelty. This behavioral pattern, characterized by heightened exploratory activity in novel environments, also suggests that early immune stimulation may contribute to the development of impulsive behavior in adolescent rats. (C) 2010 Elsevier B.V. All rights reserved.

Constituents of glandular trichomes of Tithonia diversifolia: Relationships to herbivory and antifeedant activity

The herbivory activity of the bordered patch larvae (Chlosyne lacinia, Lepidoptera) on leaves of a Brazilian population of Tithonia diversifolia and the antifeedant potential of its leaf rinse extract were investigated. The caterpillars fed only on the adaxial face, where the density of glandular trichomes is very low, and avoided the abaxial face, which contains high levels of trichomes. Deterrent activity against the larvae was observed in leaf discs treated with leaf rinse extract at concentrations of 1-5% of fresh leaf weight. High-performance liquid chromatography (HPLC) analysis indicated that sesquiterpene lactones are the main constituents of the glandular trichomes. Dichloromethane rinse extracts of the leaves and inflorescences were chemically investigated, and 16 compounds were isolated and identified: 14 sesquiterpene lactones, a flavonoid and a diterpenoid. In this study, five sesquiterpene lactones are described for the first time in the genus, including two lactones, one of which has an unusual seco-guaianolide skeleton. Our findings indicate that the caterpillars avoid the sesquiterpene-lactone-rich glandular trichomes, and provide evidence for the antifeedant activity of the dichloromethane leaf rinse extract. In addition, a study of the seasonal variation of the main constituents from the leaf surface throughout a year demonstrated that a very low qualitative but a very high quantitative variation occurs. The highest level of the main metabolite tagitinin C was observed between September and October and the lowest was from March to June, the later corresponding to the period of highest infestation by the larvae. (C) 2008 Elsevier Ltd. All rights reserved.

Study of acetylcholinesterase activity in rectal suction biopsy for diagnosis of intestinal dysganglionoses: 17-year experience of a single center

Although the utility of the acetylcholinesterase (AChE) histochemistry on rectal suction biopsy in diagnosing Hirschsprung`s disease (HD) has been documented, few reports address a great number of biopsies and patients. Our aim is to present a 17-year experience on the method of rectal suction biopsy and AChE histochemical staining for diagnosis of intestinal dysganglionoses. Between August 1989 and July 2006, 297 children suspected of having HD were submitted to rectal suction biopsies that were evaluated by the same two surgeons. There were 18 complications (6.0%), namely one self-limited rectal bleeding and 17 (5.7%) inadequate procedures that were repeated. A total of 157 patients (52.8%) showed no increased AChE activity and the remaining patients (140-47.2.0%) presented patterns of increased AChE activity confirming the diagnosis of HD or neuronal intestinal dysplasia. Among the 140 cases suspected as having HD, in 131 children the diagnosis of HD was confirmed and they were operated on. The histological studies showed that 111 children presented the classic form of HD or a long spastic segment. Sixteen children presented total colonic aganglionosis and four children proved to have intestinal neuronal dysplasia, according to histological and radiological criteria. Nine (6.6%) newborns were identified as false-positives and no false-negative results were verified. The rectal suction biopsy combined with AChE staining is advantageous for the differentiation between normal bowel and intestinal dysganglionoses. The rectal suction method is simple and can easily be performed by experienced surgeons. The histological evaluation is very objective and can be performed by a non-pathologist.

PHRENIC NERVE TRANSFER IN THE RESTORATION OF ELBOW FLEXION IN BRACHIAL PLEXUS AVULSION INJURIES: HOW EFFECTIVE AND SAFE IS IT?

OBJECTIVE: Phrenic nerve transfer has been used for treating lesions of the brachial plexus since 1970. Although, today, surgeons are more experienced with the technique, there are still widespread concerns about its effects on pulmonary function. This study was undertaken to evaluate the effectiveness and safety of this procedure. METHODS: Fourteen patients with complete palsy of the upper limb were submitted to phrenic nerve transfer as part of a strategy for surgical reconstruction of their plexuses. Two patients were lost to follow-up, and 2 patients were followed for less than 2 years. Of the remaining 10 patients, 9 (90%) were male. The lesions affected both sides equally. The mean age of the patients was 24.8 years (range, 14-43 years), and the mean interval from injury to surgery was 6 months (range, 3-9 months). The phrenic nerve was always transferred to the musculocutaneous nerve, and a nerve graft (mean length, 8 cm; range, 4.5-12 cm) was necessary in all cases. RESULTS: There was no major complication related to the surgery. Seven patients (70%) recovered functional level biceps strength (Medical Research Council grade >= 3). All of the patients exhibited a transient decrease in pulmonary function tests, but without clinical respiratory problems. CONCLUSION: On the basis of our small series and data from the literature, we conclude that phrenic nerve transfer in well-selected patients is a safe and effective procedure for recovering biceps function.

5 Hz repetitive TMS increases anticipatory motor activity in the human cortex

In the present study, we analyzed how high-frequency repetitive transcranial magnetic stimulation (rTMS) of the primary motor hand area (M1-Hand) shapes anticipatory motor activity in frontal areas as indexed by the contingent negative variation (CNV). Eight right-handed volunteers received real or sham 5 Hz rTMS at an intensity of 90% resting motorthreshold (1500 stimuli per session). Real but not sham rTMS to left M1-Hand induced a site-specific increase in amplitude of the late component of the CNV at the electrode C3 overlaying the site of stimulation. The increase in pre-movement activity in the stimulated cortex may reflect an increase in facilitatory drive from connected motor areas, enhanced responsiveness of the stimulated cortex to these inputs or both. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Attentional modulation of visual cortex activity following right parietal damage in humans

Pathological inattention following parietal damage causes perceptual impairments for visual stimuli in the contralesional hemifield. Here we used functional magnetic resonance imaging (fMRI) to examine visual cortex activity in parietal patients as they performed a spatial attention task. Righthemisphere patients and healthy controls viewed counterphasing checkerboards in which coloured targets appeared briefly within the contralesional and ipsilesional hemifields. In separate fMRI runs participants focused their attention covertiy on the left or right hemifield, or on both hemifields concurrentiy. They were required to detect coloured targets that appeared briefly within the attended hemifield(s), and to withhold responses to distractor stimuli. Neural activit}' was significantly attenuated in early visual areas within the damaged hemisphere. Crucially, although attention significantiy modulated early visual activit}' within the intact (left) hemisphere, there was relatively littie modulation of activity within the affected hemisphere. Our findings suggest that parietal lesions alter early cortical responses to contralesional visual inputs.

Premovement activity of the pre-supplementary motor area and the readiness for action: Studies of time-resolved event-related functional MRI

The supplementary motor area (SMA) is thought to play in important role in the preparation and organisation of voluntary movement. It has long been known that cortical activity begins to increase up to 2 s prior to voluntary self-initiated movement. This increasing premovement activity measured in EEG is known as the Bereitschaftspotential or readiness potential. Modern functional brain imaging methods, using event-related and time-resolved functional MRI techniques, are beginning to reveal the role of the SMA, and in particular the more anterior pre-SMA, in premovement activity associated with the readiness for action. In this paper we review recent studies using event-related time-resolved fMRI methods to examine the time-course of activation changes within the SMA throughout the preparation, readiness and execution of action. These studies suggest that the preSMA plays a common role in encoding or representing actions prior to our own voluntary self-initiated movements, during motor imagery, and from the observation of others' actions. We suggest that the pre-SMA generates and encodes motor representations which are then maintained in readiness for action. (c) 2005 Elsevier B.V. All rights reserved.

Promoting physical activity to older adults: a preliminary evaluation of three general practice-based strategies

The aim of this study was to explore the feasibility of an exercise scientist (ES) working in general practice to promote physical activity (PA) to 55 to 70 year old adults. Participants were randomised into one of three groups: either brief verbal and written advice from a general practitioner (GP) (G1, N=9); or individualised counselling and follow-up telephone calls from an ES, either with (G3, N=8) or without a pedometer (G2, N=11). PA levels were assessed at week 1, after the 12-wk intervention and again at 24 weeks. After the 12-wk intervention, the average increase in PA was 116 (SD=237) min/wk; N=28, p < 0.001. Although there were no statistically significant between-group differences, the average increases in PA among G2 and G3 participants were 195 (SD=207) and 138 (SD=315) min/wk respectively, compared with no change (0.36, SD=157) in G1. After 24 weeks, average PA levels remained 56 (SD=129) min/wk higher than in week 1. The small numbers of participants in this feasibility study limit the power to detect significant differences between groups, but it would appear that individualised counselling and follow-up contact from an ES, with or without a pedometer, can result in substantial changes in PA levels. A larger study is now planned to confirm these findings.

Antioxidant Activity of Uruguayan Propolis. In Vitro and Cellular Assays

The antioxidant capacity of propolis from the southern region of Uruguay was evaluated using in vitro as well as cellular assays. Free radical scavenging capacity was assessed by ORAC, obtaining values significantly higher than those of other natural products (8000 mu mol Trolox equiv/g propolis). ORAC values correlated well with total polyphenol content (determined by Folin-Ciocalteu method) and UV absorption. Total polyphenol content (150 mg gallic acid equiv/g propolis) and flavonoids (45 mg quercetin equiv/g propolis) were similar to values reported for southern Brazilian (group 3) and Argentinean propolis. Flavonoid composition determined by RP-HPLC indicates a strong poplar-tree origin. Samples high in polyphenols efficiently inhibit low-density lipoprotein lipoperoxidation and tyrosine nitration. In addition, Uruguayan propolis was found to induce the expression of endothelial nitric oxide synthase and inhibit endothelial NADPH oxidase, suggesting a potential cardiovascular benefit by increasing nitric oxide bioavailability in the endothelium.