Nitrosyl ethylenediaminetetraacetate ruthenium(II) complex promotes cellular growth and could be used as nitric oxide donor in plants

Over recent years nitric oxide (NO) not only has appeared as an important endogenous signaling molecule in plants and as a mediator in many developmental and physiological processes, but has also received recognition as a plant hormone. The impressive recent achievements in elucidating the role of NO in plants have come about by the application of NO donors. The aim herein was to study the effects of the different NO donors, sodium nitroprusside (SNP) and the nitrosyl ethylenediaminetetraacetate ruthenium(II) ([Ru(NO)(Hedta)]) complex on cellular growth in embryogenic suspension cultures of Araucaria angustifolia. Appraisal of our data revealed that [Ru(NO)(Hedta)] stimulated about 60% of cellular growth in embryogenic suspension cultures of A. angustifolia, with results similar to those observed with the SNP donor. Nevertheless, application of the NO scavenger PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) inhibited this cellular growth in both. Cellular growth was correlated with an increase in endogenous NO levels after 21 days of culture, especially in treatments with NO donors. Our results demonstrated that the [Ru(NO)Hedta] complex could possibly be used as a NO donor in plants. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Expression of Circadian Clock and Melatonin Receptors within Cultured Rat Cardiomyocytes

Melatonin, the pineal gland hormone, provides entrainment of many circadian rhythms to the ambient light/dark cycle. Recently, cardiovascular studies have demostrated melatonin interactions with many physiological processes and diseases, such as hypertension and cardiopathologies. Although membrane melatonin receptors (MT1, MT2) and the transcriptional factor ROR alpha have been reported to be expressed in the heart, there is no evidence of the cell-type expressing receptors as well as the possible role of melatonin on the expression of the circadian clock of cardiomyocytes, which play an important role in cardiac metabolism and function. Therefore, the aim of this study was to evaluate the mRNA and protein expressions of MT1, MT2, and ROR alpha and to determine whether melatonin directly influences expression of circadian clocks within cultured rat cardiomyocytes. Adult rat cardiomyocyte cultures were created, and the cells were stimulated with 1 nM melatonin or vehicle. Gene expressions were assayed by real-time polymerase chain reaction (PCR). The mRNA and protein expressions of membrane melatonin receptors and RORa were established within adult rat cardiomyocytes. Two hours of melatonin stimulation did not alter the expression pattern of the analyzed genes. However, given at the proper time, melatonin kept Rev-erb alpha expression chronically high, specifically 12 h after melatonin treatment, avoiding the rhythmic decline of Rev-erb alpha mRNA. The blockage of MT1 and MT2 by luzindole did not alter the observed melatonin-induced expression of Rev-erb alpha mRNA, suggesting the nonparticipation of MT1 and MT2 on the melatonin effect within cardiomyocytes. It is possible to speculate that melatonin, in adult rat cardiomyocytes, may play an important role in the light signal transduction to peripheral organs, such as the heart, modulating its intrinsic rhythmicity. (Author correspondence: cipolla@icb.usp.br)

Mechanism of acetylcholine-induced calcium signaling during neuronal differentiation of P19 embryonal carcinoma cells in vitro

Muscarinic (mAChRs) and nicotinic acetylcholine receptors (nAChRs) are involved in various physiological processes, including neuronal development. We provide evidence for expression of functional nicotinic and muscarinic receptors during differentiation of P19 carcinoma embryonic cells, as an in vitro model of early neurogenesis. We have detected expression and activity alpha(2)-alpha(7), beta(2), beta(4) nAChR and M1-M5 mAChR subtypes during neuronal differentiation. Nicotinic alpha(3) and beta(2) mRNA transcription was induced by addition of retinoic acid to P19 cells. Gene expression Of alpha(2), alpha(4)-alpha(7), beta(4) nAChR subunits decreased during initial differentiation and increased again when P19 cells underwent final maturation. Receptor response in terms of nicotinic agonist-evoked Ca2+, flux was observed in embryonic and neuronal-differentiated cells. Muscarinic receptor response, merely present in undifferentiated P19 cells, increased during neuronal differentiation. The nAChR-induced elevation of intracellular calcium ([Ca2+](i)) response in undifferentiated cells was due to Ca2+ influx. In differentiated P19 neurons the nAChR-induced [Ca2+](i) response was reduced following pretreatment with ryanodine, while the mAChR-induced response was unaffected indicating the contribution of Ca2+ release from ryanodine-sensitive stores to nAChR- but not mAChR-mediated Ca2+ responses. The presence of functional nAChRs in embryonic cells suggests that these receptors are involved in triggering Ca2+ waves during initial neuronal differentiation. (C) 2007 Elsevier Ltd. All rights reserved.

Insulin modulates norepinephrine-mediated melatonin synthesis in cultured rat pineal gland

The mammalian pineal gland synthesizes melatonin in a circadian manner, peaking during the dark phase. This synthesis is primarily regulated by sympathetic innervations via noradrenergic fibers, but is also modulated by many peptidergic and hormonal systems. A growing number of studies reveal a complex role for melatonin in influencing various physiological processes, including modulation of insulin secretion and action. In contrast, a role for insulin as a modulator of mclatonin synthesis has not been investigated previously. The aim of the current study was to determine whether insulin modulates norepinephrine (NE)-mediated melatonin synthesis. The results demonstrate that insulin (10(-8)M) potentiated norepinephrine-mediated melatonin synthesis and tryptophan hydroxylase (TPOH) activity in ex vivo incubated pineal glands. When ex vivo incubated pineal glands were synchronized (12h NE-stimulation, followed by 12h incubation in the absence of NE), insulin potentiated NE-mediated melatonin synthesis and arylalkylamine-N-acetyltransferase (AANAT) activity. Insulin did not affect the activity of hydroxyindole-O-methyltranferase (HIOMT), nor the gene expression of tpoh, aanat, or hiomt, under any of the conditions investigated. We conclude that insulin potentiates NE-mediated melatonin synthesis in cultured rat pineal gland, potentially through post-transcriptional events. (C) 2007 Elsevier Inc. All rights reserved.

REACTIVE OXYGEN SPECIES AND THE STRUCTURAL REMODELING OF THE VISUAL SYSTEM AFTER OCULAR ENUCLEATION

Redox processes associated with controlled generation of reactive oxygen species (ROS) by NADPH oxidase (Nox) add an essential level of regulation to signaling pathways underlying physiological processes. We evaluated the ROS generation in the main visual relays of the mammalian brain, namely the superior colliculus (SC) and the dorsal lateral geniculate nucleus (DLG), after ocular enucleation in adult rats. Dihydroethidium (DHE) oxidation revealed increased ROS generation in SC and DLG between 1 and 30 days postlesion. ROS generation was decreased by the Nox inhibitors diphenyleneiodonium chloride (DPI) and apocynin. Real-time PCR results revealed that Nox 2 was upregulated in both retinorecipient structures after deafferentation, whereas Nox 1 and Nox 4 were upregulated only in the SC. To evaluate the role of ROS in structural remodeling after the lesions, apocynin was given to enucleated rats and immunohistochemistry was conducted for markers of neuronal remodeling into SC and DLG. Immunohistochemical data showed that ocular enucleation produces an increase of neurofilament and microtubule-associated protein-2 immunostaining in both SC and DLG, which was markedly attenuated by apocynin treatment. Taken together, the findings of the present study suggest a novel role for Nox-induced ROS signaling in mediating neuronal remodeling in visual areas after ocular enucleation. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Effects of Plasmodium gallinaceum on hemolymph physiology of Aedes aegypti during parasite development

Insect disease vectors show diminished fecundity when infected with Plasmodium. This phenomenon has already been demonstrated in laboratory models such as Aedes aegypti, Anopheles gambiae and Anopheles stephensi. This study demonstrates several changes in physiological processes of A. aegypti occurring upon infection with Plasmodium gallinaceum, such as reduced ecdysteroid levels in hemolymph as well as altered expression patterns for genes involved in vitellogenesis, lipid transport and immune response. Furthermore, we could show that P. gallinaceum infected A. aegypti presented a reduction in reproductive fitness, accompanied by an activated innate immune response and increase in lipophorin expression, with the latter possibly representing a nutritional resource for Plasmodium sporozoites. (C) 2010 Elsevier Ltd. All rights reserved.

Coupling Virus-Induced Gene Silencing to Exogenous Green Fluorescence Protein Expression Provides a Highly Efficient System for Functional Genomics in Arabidopsis and across All Stages of Tomato Fruit Development

Since the advent of the postgenomic era, efforts have focused on the development of rapid strategies for annotating plant genes of unknown function. Given its simplicity and rapidity, virus-induced gene silencing (VIGS) has become one of the preeminent approaches for functional analyses. However, several problems remain intrinsic to the use of such a strategy in the study of both metabolic and developmental processes. The most prominent of these is the commonly observed phenomenon of ""sectoring"" the tissue regions that are not effectively targeted by VIGS. To better discriminate these sectors, an effective marker system displaying minimal secondary effects is a prerequisite. Utilizing a VIGS system based on the tobacco rattle virus vector, we here studied the effect of silencing the endogenous phytoene desaturase gene (pds) and the expression and subsequent silencing of the exogenous green fluorescence protein (gfp) on the metabolism of Arabidopsis (Arabidopsis thaliana) leaves and tomato (Solanum lycopersicum) fruits. In leaves, we observed dramatic effects on primary carbon and pigment metabolism associated with the photobleached phenotype following the silencing of the endogenous pds gene. However, relatively few pleiotropic effects on carbon metabolism were observed in tomato fruits when pds expression was inhibited. VIGS coupled to gfp constitutive expression revealed no significant metabolic alterations after triggering of silencing in Arabidopsis leaves and a mild effect in mature green tomato fruits. By contrast, a wider impact on metabolism was observed in ripe fruits. Silencing experiments with an endogenous target gene of interest clearly demonstrated the feasibility of cosilencing in this system; however, carefully constructed control experiments are a prerequisite to prevent erroneous interpretation.

Basic oxygen furnace slag as a treatment material for pathogens: Contribution of inactivation and attachment in virus attenuation

Basic oxygen furnace (BOF) slag media were studied as a potential treatment material in on-site sanitation systems. Batch and column studies were conducted to evaluate attenuation of the bacteriophage PR772 and 0.190 mu m diameter microspheres by BOF media, and to delineate the relative contributions of two principle processes of virus attenuation: inactivation and attachment. In the batch studies, conducted at 4 degrees C, substantial inactivation of PR772 did not occur in the pH 7.6 and 9.5 suspensions. At pH 11.4, bimodal inactivation of PR772 was observed, at an initial rate of 2.1 log C/C(0) day(-1) for the first two days, followed by a much slower rate of 0.124 log C/C(0) day(-1) over the following 10 days. Two column studies were conducted at 4 degrees C at a flow rate of 1 pore volume day(-1) using two slag sources (Stelco, Ontario; Tubarao, Brazil) combined with sand and pea gravel. In both column experiments, the effluent microsphere concentration approached input concentrations over time (reductions of 0.1-0.2 log C/C(0)), suggesting attachment processes for microspheres were negligible. Removal of PR772 virus was more pronounced both during the early stages of the experiments, but also after longer transport times (0.5-1.0 log C/C(0)). PR772 reduction appeared to be primarily as a result of virus inactivation in response to the elevated pH conditions generated by the BOF mixture (10.6-11.4). On-site sanitation systems using BOF media should be designed to maintain sufficient contact time between the BOF media and the wastewater to allow sufficient residence time of pathogens at elevated pH conditions. (C) 2009 Published by Elsevier Ltd.